CN103232539B - Antibodies to opgl - Google Patents

Abstract

Antibodies that interact with osteoprotegerin ligand (OPGL) are described. Methods of treating osteopenic disorders by administering a pharmaceutically effective amount of antibodies to OPGL are described. Methods of detecting the amount of OPGL in a sample using antibodies to OPGL are described.

Description

anti-OPGL antibody

This application is a divisional application of the Chinese patent application 02816680.9 "anti-OPGL antibody" filed on June 25, 2002.

This application claims priority to US Provisional Application Serial No. 60/301,172, filed June 26, 2001, which is hereby incorporated by reference for any purpose.

technical field

The present invention relates to antibodies that bind to osteoprotegerin ligand (OPGL). Compositions and methods for treating bone disorders such as osteoporosis, arthritic bone loss, Paget's disease, and osteopenia are also described.

Background technique

Bone tissue provides support for the body and includes minerals (including calcium and phosphorus), a collagenous and non-collagenous matrix, and cells. Living tissue exhibits a dynamic balance between bone formation known as deposition and bone breakdown known as resorption. All three types of cells found in bone, osteocytes, osteoblasts and osteoclasts, are involved in this balance. Osteoblasts promote the formation of bone tissue, while osteoclasts are involved in resorption. Resorption or dissolution of bone matrix and minerals is a rapid and efficient process compared to bone formation and releases large amounts of minerals from bone. Osteoclasts are involved in the regulation of normal remodeling of bone tissue and hormone-induced resorption. For example, secretion of parathyroid hormone in response to reduced concentrations of calcium ions in the extracellular fluid stimulates reabsorption. Rather, inhibition of reuptake is a function of calcitonin. In addition, metabolites of vitamin D alter the bone response to parathyroid hormone and calcitonin.

Osteoprotegerin ligand (OPGL), a member of the TNF family of cytokines, promotes osteoclastosis by binding to a receptor activator of NF-κB (RANK, also known as osteoclast differentiation and activation receptor, or ODAR) Cell formation. Osteoprotegerin (OPG), on the other hand, inhibits osteoclast formation by sequestering OPGL and preventing the association of OPGL with ODARs. Thus, the amount of OPGL associated with ODARs correlates with the balance between bone deposition and resorption.

After skeletal maturation, the amount of bone in the skeleton reflects the balance (or imbalance) of bone formation and bone resorption. Bone mass peaks before the fourth decade after skeletal maturity. Between the fourth and fifth decade, the balance shifts and bone resorption predominates. The inevitable decline in bone mass with age begins earlier in women than in men and accelerates markedly after menopause in some women (mainly those of Caucasian and Asian ancestry).

Osteopenia is a condition associated with any decrease in general bone mass below normal. Such a condition can be a condition that results from a slowed rate of bone synthesis, or an increased rate of bone destruction, or both. A common form of osteopenia is primary osteoporosis, also known as postmenopausal and senile osteoporosis. This form of osteoporosis is the result of general bone loss with age, and often the result of accelerated bone resorption that accompanies the normal rate of bone formation. Many white women in the United States develop symptomatic osteoporosis. A direct relationship exists between osteoporosis and the incidence of hip, femoral, cervical, and intertrochanteric fractures in women 45 years and older. Symptomatic osteoporosis may develop in older men between the ages of 50 and 70. In certain instances, osteoporosis may result from increased levels or activity of OPGL. Accordingly, molecules that modulate the activity of OPGL in osteoclastogenesis are useful.

Several factors have been identified that contribute to postmenopausal and senile osteoporosis. They include changes in hormone levels that accompany aging and inappropriate calcium consumption due to decreased intestinal absorption of calcium and other minerals. Some treatments include hormone therapy or dietary supplements to delay the process. More recently, antiresorptive agents such as bisphosphonates and selective estrogen receptor modifiers (SERMs) have been used to prevent and treat reduced bone mass. Therefore, combining those treatments with molecules that modulate OPGL activity would be useful in the treatment of certain osteopenic diseases.

Contents of the invention

In some embodiments, the present invention provides an antibody comprising a heavy chain and a light chain, wherein the heavy chain includes the amino acid sequence shown in SEQ ID NO: 2 or a fragment thereof, and the light chain includes the amino acid sequence shown in SEQ ID NO: 4 or fragments thereof.

In some embodiments, the present invention provides an antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises a variable region comprising the amino acid sequence shown in SEQ ID NO: 13 or a fragment thereof, wherein the light chain comprises a variable region comprising SEQ ID NO: 13 The variable region of the amino acid sequence shown in NO: 14 or a fragment thereof.

In some embodiments, the invention provides an antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence shown in SEQ ID NO: 2 or a fragment thereof.

In some embodiments, the invention provides an antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises a variable region comprising the amino acid sequence shown in SEQ ID NO: 13 or a fragment thereof.

In some embodiments, the invention provides an antibody comprising a heavy chain and a light chain, wherein the light chain comprises the amino acid sequence shown in SEQ ID NO: 4 or a fragment thereof.

In some embodiments, the invention provides an antibody comprising a heavy chain and a light chain, wherein the light chain comprises a variable region comprising the amino acid sequence shown in SEQ ID NO: 14 or a fragment thereof.

In some embodiments, the invention provides an antibody comprising a heavy chain and a light chain, (a) wherein the heavy chain comprises a first variable region, wherein the first variable region comprises the amino acid set forth in SEQ ID NO: 13 a sequence having at least 90% identity to the sequence, and (b) wherein the light chain comprises a second variable region, wherein the second variable region comprises a sequence having at least 90% identity to the amino acid sequence shown in SEQ ID NO: 14 , and (c) wherein the antibody interacts with osteoprotegerin ligand (OPGL).

In some embodiments, the first variable region comprises a sequence having at least 95% identity to the amino acid sequence set forth in SEQ ID NO: 13, and the second variable region comprises a sequence having at least 95% identity to the amino acid sequence set forth in SEQ ID NO: 14 Sequences that are at least 95% identical.

In some embodiments, the first variable domain comprises a sequence at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 13, and the second variable domain comprises a sequence having at least 99% identity to the amino acid sequence set forth in SEQ ID NO: 14. Sequences that are at least 99% identical.

In some embodiments, the invention provides a heavy chain comprising the amino acid sequence shown in SEQ ID NO: 2 or a fragment thereof. In some embodiments, the present invention provides a heavy chain comprising a variable region and a constant region, wherein the variable region comprises the amino acid sequence shown in SEQ ID NO: 13 or a fragment thereof.

In some embodiments, the invention provides a light chain comprising the amino acid sequence shown in SEQ ID NO: 4 or a fragment thereof. In some embodiments, the invention provides a light chain comprising the amino acid sequence shown in SEQ ID NO: 14 or a fragment thereof.

In some embodiments of the invention, single chain antibodies are provided. In some embodiments of the invention, single chain Fv antibodies are provided. In some embodiments of the invention, Fab antibodies are provided. In some embodiments of the invention, Fab' antibodies are provided. In some embodiments of the invention, (Fab') ₂ antibodies are provided.

In some embodiments, pharmaceutical compositions containing antibodies of the invention are provided. In some embodiments, pharmaceutical compositions comprising a therapeutically effective amount of an anti-OPGL antibody are provided.

In some embodiments, the pharmaceutical composition contains an anti-OPGL antibody and at least one therapeutic agent selected from the group consisting of bone morphogenetic factor, transforming growth factor-beta (TGF-beta), interleukin-1 (IL-1 ) inhibitors, IL-1ra, Kineret ^TM , anakinra, TNFα inhibitors, soluble TNFα receptors, Enbrel ^TM , etanercept, anti-TNFα antibody, Remicade ^TM , infliximab, D2E7 antibody, parathyroid hormone, parathyroid Adrenaline analogues, parathyroid hormone-related protein, parathyroid hormone-related protein analogues, prostaglandins, bisphosphonates, alendronate, fluoride, calcium, non-steroidal anti-inflammatory drugs (NSAIDs), COX- 2 Inhibitors, Celebrex ^™ , Celecoxib, Vioxx ^™ , Rofixib, Immunosuppressants, Methotrexate, Leflunomide, Serine Protease Inhibitors, Secretory Leukocyte Protease Inhibitors (SLPI), IL -6 inhibitor, anti-IL-6 antibody, IL-8 inhibitor, anti-IL-8 antibody, IL-18 inhibitor, IL-18 binding protein, anti-IL-18 antibody, interleukin-1 converting enzyme (ICE) regulation agent, fibroblast growth factor (FGF), FGF modulator, PAF antagonist, keratinocyte growth factor (KGF), KGF-related molecule, KGF modulator; matrix metalloproteinase (MMP) modulator, nitric oxide synthase (NOS) modulators, glucocorticoid receptor modulators, glutamate receptor modulators, lipopolysaccharide (LPS) level modulators, norepinephrine, norepinephrine mimetics, and norepinephrine modulators .

In some embodiments of the present invention, there is provided a method of treating an osteopenic disease comprising administering a pharmaceutically effective amount of an antibody. In some embodiments, a method of treating an osteopenic disease comprising administering a pharmaceutical composition is provided.

In some embodiments, there is provided a method of treating an inflammatory condition with bone loss in a patient comprising administering a pharmaceutical composition.

In some embodiments, there is provided a method of treating an autoimmune disease with bone loss in a patient comprising administering a pharmaceutical composition.

In some embodiments, there is provided a method of treating rheumatoid arthritis in a patient comprising administering a pharmaceutical composition of the invention.

In an embodiment of the invention there is provided a method of detecting the level of OPGL in a biological sample comprising contacting the sample with an antibody.

Description of drawings

Figure 1 shows the cDNA sequence (SEQ ID NO: 1) encoding the heavy chain of the αOPGL-1 antibody. Specifically described the DNA sequence of the heavy chain expression plasmid from the HindIII site to the SalI site, the start codon starts at nt14, and the stop codon starts at nt1415.

Figure 2 shows the amino acid sequence (SEQ ID NO: 2) of the heavy chain of the αOPGL-1 antibody. Among them, the IgG2 signal peptide is underlined, the variable region is capitalized but not underlined, and the constant region is lowercased.

Figure 3 shows the cDNA sequence (SEQ ID NO: 3) encoding the light chain of the αOPGL-1 antibody. Specifically described the DNA sequence of the K chain expression plasmid sequence from the Xba I site to the Sal I site. The start codon starts at nt12; the stop codon starts at nt717.

Figure 4 shows the amino acid sequence (SEQ ID NO: 4) of the light chain of the αOPGL-1 antibody. Among them, the K signal peptide is underlined, the variable region is capitalized but not underlined, and the constant region is lowercase.

Fig. 5 shows a schematic diagram of αOPGL-1κ light chain expression plasmid αOPGL-1-κ/pDSRa19.

Figure 6 shows a schematic diagram of the αOPGL-1 IgG2 heavy chain expression plasmid, αOPGL-1-IgG2/pDSRa19.

Figure 7 shows the dose-dependent binding of [alpha]OPGL-1 to OPGL-coated EIA plates.

αOPGL-1 binds to soluble OPG ligands coated on EIA plates in a dose-dependent manner.

The measurement method is:

96-well EIA plates were coated with recombinant soluble OPGL. Different concentrations of αOPGL-1 were added to the wells and incubated at room temperature for approximately 2 hours. Binding was detected with goat anti-human IgG (Fab')-horseradish peroxidase Antibody. Read absorbance at 450nm and 650nm.

Figure 8 shows the specific binding of [alpha]OPGL-1 to membrane-bound OPGL.

αOPGL-1 binds to OPGL expressed on the cell surface of transfected CHO REN218-9 cells in a dose-dependent manner. This binding competes with exogenously added human OPGL but not with TNF-α, TNF-β, TRAIL or CD40 Ligand competition. Pre-incubation of αOPGL-1 (100ng/ml) with various concentrations of soluble OPGL or other ligands, followed by incubation with CHO REN218-9 cells expressing OPGL on the surface. Cells were then incubated at 2-8°C Incubate with FITC-labeled F(ab') ₂ goat anti-human IgG, Fcr fragment-specific for 30 minutes. After centrifugation and washing the cell surface, use flow cytometry to measure.

Figure 9 shows the inhibitory effect of soluble OPGL on [alpha]OPGL-1 binding to OPGL-coated EIA plates. Exogenously added soluble OPGL competitively reduces the binding of αOPGL-1 to OPGL on EIA plates.

Figure 10 shows the specific binding of αOPGL-1 to OPGL-coated EIA plates.

αOPGL-1 does not bind TNF family members TNFα, TNFβ, TRAIL or CD40 ligands.

Exogenously added TNF-α, TNF-β, TRAIL or CD40 ligands did not reduce the binding of αOPGL-1 to OPGL on EIA plates.

Figure 11 shows the dose-dependent inhibitory effect of αOPGL-1 on osteoclastogenesis. Specifically showing a dose-dependent inhibitory effect of αOPGL-1 on OPG ligand-induced TRAP activity in naive 264.7 cells.

Figure 12 shows the dose-dependent inhibition of OPGL binding to ODAR by [alpha]OPGL-1. Specifically, a dose-dependent inhibitory effect of [alpha]OPGL-1 on the binding of europium-labeled OPG ligand to OPAR-FLAG/anti-FLAG-APC is shown.

Figure 13 shows mean serum concentration-time profiles following administration of a single dose of αOPGL-1 to rhesus monkeys. Specific display:

Mean (±SD) serum-concentration time profiles following single dose administration of αOPGL-1 to rhesus monkeys at doses of 0.1, 1 and 10.0 mg/kg IV (n=2/dose) and 1.0 mg/kg SC (n=6/dose).

Figure 14 shows the mean percent change in serum N-Tx concentration after administration of a single dose of αOPGL-1 to rhesus monkeys. Specific display:

A single dose IV (n=2/dose) or

Mean (±SD) percent change in serum N-TX concentrations following SC (n=6) administration of αOPGL-1.

Figure 15 shows the mean percent change in urinary N-Tx concentration after administration of a single dose of αOPGL-1 to rhesus monkeys. Specific display:

A single dose IV (n=2/dose) or

Mean (±SD) percent change in urine N-TX concentration after SC (n=6) administration of αOPGL-1.

Figure 16 shows antibody positive and negative serum-concentration time profiles following administration of a single dose of αOPGL-1 to rhesus monkeys. Specific display:

Cynomolgus monkeys were administered a single dose of αOPGL-1 IV at a dose of 0.1 mg/kg

Antibody-positive (open symbols) and negative (closed symbols) animal serum-concentration-time curves thereafter.

Figure 17 shows the amino acid sequence (SEQ ID NO: 13) of the heavy chain variable region of the αOPGL-1 antibody.

Figure 18 shows the amino acid sequence (SEQ ID NO: 13) of the light chain variable region of the αOPGL-1 antibody.

Fig. 19 shows a cell culture method for producing αOPGL-1.

Figure 20 shows the mean percent change in serum calcium from baseline following administration of a single dose of aOPGL-1 to macaques.

Figure 21 shows the mean percent change in serum alkaline phosphatase from baseline following administration of a single dose of [alpha]OPGL-1 to rhesus monkeys.

Detailed ways

The headings used here are for organizational purposes and should not be construed as limiting the subject matter described. All references cited in this application are expressly incorporated herein by reference for any purpose.

definition

Standard techniques can be used for recombinant DNA, oligonucleotide synthesis, and tissue culture and transformation (eg, electroporation, lipofection). Enzymatic reactions and purification techniques can be performed according to manufacturer's instructions or according to conventional techniques in the art or as described herein. The techniques and methods described above can generally be performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification. See, eg, Sambrook et al., Molecular Cloning: A Laboratory Manual (Second Edition, Cold Spring Harbor Experimental Press, Cold Spring Harbor, New York (1989)), which is incorporated herein by reference for any purpose. Except where specific definitions are indicated, the relative terminology used, analytical chemistry, synthetic organic chemistry, and medical and pharmaceutical chemistry, and laboratory methods and techniques described herein are those well known and commonly used in the art. Standard techniques can be used for chemical syntheses, chemical analyses, pharmaceutical preparation, formulation and delivery, and treatment of patients.

As used in the present disclosure, the following terms, unless otherwise stated, should be understood to have the following definitions:

The term "isolated polynucleotide" as used herein shall refer to genomic polynucleotide, cDNA, or of synthetic origin or a combination thereof, depending on its source, "isolated polynucleotide" (1) and "isolated polynucleotide "is a polynucleotide found in nature in whole or in part without association, (2) linked to a polynucleotide that is not associated with one in nature, or (3) as part of a larger sequence that does not occur in nature.

The term "isolated protein" herein refers to a protein encoded by a cDNA, recombinant RNA, or synthetic source, or a combination thereof, that (1) is free of at least some of the protein normally found, (2) substantially free of the same origin, e.g., from the same species other proteins that are (3) expressed by cells from a different species, or (4) do not occur in nature.

The term "polypeptide" as used herein as a genetic term refers to a native protein, or a sequence having one or more amino acid deletions, additions and/or substitutions from the native sequence. The term "polypeptide" also includes αOPGL-1 (described below, SEQ ID NO: 2 and SEQ ID NO: 4), or a sequence of one or more amino acid deletions, additions and/or substitutions of αOPGL-1. According to some embodiments, the invention comprises a human heavy chain immunoglobulin molecule represented in Figure 2 (SEQ ID NO:2) and a human light chain immunoglobulin molecule represented in Figure 4 (SEQ ID NO:4) or a fragment or the like thereof things.

The term "naturally occurring" as used herein with respect to a substance refers to the fact that the substance can be found in nature. For example, a polypeptide or polynucleotide sequence that occurs in an organism (including a virus) that can be isolated from a natural source and has not been intentionally altered by man experimentally or otherwise is naturally occurring.

The term "operably linked" as used herein refers to elements in a relationship that permits them to function in their intended manner. For example, a regulatory sequence "operably linked" to a coding sequence is ligated in such a way that expression of the coding sequence is achieved under conditions compatible with the regulatory sequence.

The term "regulatory sequences" as used herein refers to polynucleotide sequences that enable the expression and processing of the coding sequences to which they are linked. The nature of such regulatory sequences will vary depending on the host organism. According to some embodiments, prokaryotic regulatory sequences may include promoters, ribosomal binding sites, and transcription termination sequences. According to some embodiments, eukaryotic regulatory sequences may include promoter and transcription termination sequences. In some embodiments, "regulatory sequences" may include leader sequences and/or fusion partner sequences.

The term "polynucleotide" referred to herein means a polymeric form of nucleotides of at least 10 bases in length. In some embodiments, the bases may be ribonucleotides or deoxyribonucleotides or modified forms of both types of nucleotides. The term includes both single- and double-stranded forms of DNA.

The term "oligonucleotide" as referred to herein includes naturally occurring and modified nucleotides linked together by naturally occurring and/or non-naturally occurring oligonucleotide bonds. Oligonucleotides are a subset of polynucleotides generally comprising a length of 200 bases or less. In some embodiments, the oligonucleotides are 10-60 bases in length. In some embodiments, the oligonucleotide is 12, 13, 14, 15, 16, 17, 18, 19 or 20-40 bases in length. Oligonucleotides may be single-stranded or double-stranded, for example for the construction of genetic mutants. Oligonucleotides of the invention may be sense or antisense oligonucleotides.

The term "naturally occurring nucleotides" includes deoxyribonucleotides and ribonucleotides. The term "modified nucleotide" includes nucleotides with modified or substituted sugar groups and the like. The term "oligonucleotide linkage" includes such oligonucleotide linkages as phosphorothioate, phosphorodithioate, phosphoroselenoate, phosphordiselenoate, phosphoroanilothioate, phosphoroaniladate, phosphoroamidate, and the like. See, e.g., LaPlanche et al., Nucl. Acids Res. 14:9081 (1986); Stec et al., J. Am. Chem. Soc. 106:6077 (1984); Stein et al., Nucl. Acids Res. 16:3209 (1988); Zon et al., Anti-Cancer Drug Design 6: 539 (1991); Zon et al., Oligonucleotides and Analogues: A Practical Approach, pp87-108 (F. Eckstein, Ed., Oxford University Press, Oxford England (1991)); Stec et al., U.S. Patent No. 5151510; Uhlmann and Peyman Chemical Reviews 90:543 (1990), the disclosures of which are hereby incorporated by reference for any purpose. An oligonucleotide can include a detectable label.

Identity and similarity of related polypeptides are readily calculated by well known methods. Such methods include, but are not limited to, those described in the following literature:

Computational Molecular Biology, Lesk, A.M., Ed., Oxford University Press, New York (1988); Biocomputing: Informatics and Genome Projects, Smith, D.W., Ed., Academic Press, New York (1993); Computer Analysis of Sequence Data, Part 1, Griffin, A.M., and Griffin, H.G., eds., Humana Press, New Jersey (1994); Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press (1987); Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds. , M. Stockton Press, New York (1991); and Carillo et al., SIAM J. Applied Math., 48:1073 (1988).

A preferred method for determining identity is designed to show the greatest match between the sequences analyzed. Methods for determining identity are described in publicly available computer programs. Preferred computer program methods for determining the identity between two sequences include, but are not limited to, the GCG package, including GAP (Devereux et al., Nucl. Acids Res. 12:387 (1984); Genetics Computer Group, University of Wisconsin, Madison, WI, BLASTP, BLASTN, and FASTA (Altschul et al., J. Mol. Biol., 215:403-410 (1990)). The BLASTX program is publicly available from the National Center for Biotechnology Information (NCBI) and other sources (BLAST Manual, Altschul et al., NCB/NLM/NIH Bethesda, MD 20894; Altschul et al., supra (1990)). The well-known SmithWaterman algorithm can also be used to determine identity.

Some alignment systems used to align two amino acid sequences may result in only a short region of the two sequences matching, even though there is no significant relatedness between the two full-length sequences, this small aligned region may have a very high sequence identity. Thus, in some embodiments, the selected alignment method (GAP program) will result in an alignment spanning at least 50 contiguous amino acids of the target polypeptide.

For example, using the computer algorithm GAP (Genetics Computer Group, University of Wisconsin, Madison, WI), two polypeptides whose percent sequence identity is to be determined are aligned for their respective amino acid best matches ("match span", as determined by the algorithm) . In some embodiments, the gap opening penalty (calculated as 3X average diagonal; "average diagonal" is the average of the diagonals of the comparison matrix used for the comparison; "diagonal" is the A score or number assigned to each perfect amino acid match) and a gap extension penalty (which is typically 1/10 times the gap opening penalty), and comparison matrices such as PAM250 or BLOSUM62 are used in conjunction with the algorithm. In some embodiments, the algorithm also uses standard comparison matrices (for the PAM250 comparison matrix, see Dayhoff et al., Atlas of Protein Sequence and Structure, 5(3) (1978); for the BLOSUM62 comparison matrix, see Henikoff et al., Proc. Natl. Acad . Sci USA, 89: 10915-10919 (1992)).

In some embodiments, parameters for comparison of polypeptide sequences include the following:

Algorithms: Needleman et al., J. Mol. Biol., 48:443-453 (1970);

Comparison matrix: BLOSUM62 by Henikoff et al., supra (1992);

Gap Penalty: 12

Gap Length Penalty: 4

Similarity Threshold: 0

It is useful for the GAP program to use the above parameters. In some embodiments, there are default parameters for polypeptide comparisons using the GAP algorithm (no penalty for end gaps).

As used herein, the conventional usage of the twenty common amino acids and their abbreviations are as follows. See Immunology-A Synthesis (Second Edition, E.S. Golub and D.R. Gren, Eds., Sinauer Associates, Sunderland, Mass (1991)), incorporated herein by reference for any purpose. Twenty kinds of common amino acids, unnatural amino acids such as α-, α-disubstituted amino acids, N-alkyl amino acids, lactic acid, and other stereoisomers of amino acids that are not common (such as D-amino acids) can also be polypeptides of the present invention suitable ingredients. Examples of uncommon amino acids include: 4-hydroxyproline, gamma-carboxyglutamic acid, ε-N,N,N-trimethyllysine, ε-N-acetyllysine, O-phosphate Serine, N-acetylserine, N-formylmethionine, 3-methylhistidine, 5-hydroxylysine, σ-N-methylarginine, and other similar amino acids and imino acids ( such as 4-hydroxyproline). In accordance with standard usage and convention, in polypeptide notation used herein, the left-hand direction is the amino-terminal direction and the right-hand direction is the carboxy-terminal direction.

Similarly, unless specifically stated otherwise, the left-hand end of a single-stranded polynucleotide sequence is the 5' end; the left-hand direction of a double-stranded polynucleotide sequence is referred to as the 5' direction. The direction of 5' to 3' addition of the nascent RNA transcript is called the direction of transcription; the sequence region on the DNA strand that has the same sequence as the RNA and is the 5' to 5' end of the RNA transcript is called the "upstream sequence"; The region of sequence on the DNA strand that has the same sequence as RNA and is 3' to 3' of the RNA transcript is called "downstream sequence".

Conservative amino acid substitutions may include non-naturally occurring amino acid residues that are typically inserted by chemical peptide synthesis rather than by biological systems. These include peptidomimetics and other inverted or inverted forms of amino acid moieties.

Naturally occurring residues can be divided into several groups based on common side chain properties:

1) Hydrophobicity: norleucine, Met, Ala, Val, Leu, Ile;

2) Neutral hydrophilicity: Cys, Ser, Thr, Asn, Gln;

3) Acidity: Asp, Glu;

4) Basic: His, Lys, Arg;

5) Residues affecting chain orientation: Gly, Pro; and

6) Aromatic: Trp, Tyr, Phe.

For example, non-conservative substitutions may involve exchanging a member of one of these classes for a member of another class. Such substituted residues may be introduced into regions of the human antibody that are homologous to the non-human antibody, or into nonhomologous regions of the molecule.

According to some embodiments, such changes are made taking into account the hydropathic index of the amino acid. Each amino acid is assigned a hydropathic index based on its hydrophobicity and charge characteristics. They are: Isoleucine (+4.5); Valine (+4.2); Leucine (+3.8); Phenylalanine (+2.8); Cysteine/Cystine (+2.5); Methionine (+1.9); Alanine (+1.8); Glycine (-0.4); Threonine (-0.7); Serine (-0.8); Tryptophan (-0.9); Tyrosine (- 1.3); proline (-1.6); histidine (-3.2); glutamic acid (-3.5); glutamine (-3.5); aspartic acid (-3.5); asparagine (-3.5 ); lysine (-3.9); and arginine (-4.5).

The importance of amino acid hydropathic indices in conferring interactive biological functions on proteins is well known in the art. Kyte et al., J. Mol. Biol. 157:105-131 (1982). It is known that some amino acids can be substituted for other amino acids with similar hydropathic indices or scores and still retain similar biological activity. Alterations are made on the basis of hydropathic index, and in some embodiments include substitution of amino acids whose hydropathic index is within ±2. In some embodiments, include those whose hydropathic index is within ±1, and in some embodiments, include those whose hydropathic index is within ±0.5.

It is also known in the art that substitutions of similar amino acids can be efficiently made on the basis of hydrophilicity, particularly where the biologically functional protein or peptide so produced is intended for use in immunization embodiments, as in the case of the present application. In some embodiments, the maximum local average hydrophilicity of a protein, as governed by the hydrophilicity of its neighboring amino acids, correlates with its immunogenicity and antigenicity, ie correlates with the biological properties of the protein.

The following hydrophilicity values were assigned to these amino acid residues: arginine (+3.0); lysine (+3.0); aspartic acid (+3.0±1); glutamic acid (+3.0±1); Serine (+0.3); Asparagine (+0.2); Glutamine (+0.2); Glycine (0); Threonine (-0.4); Proline (-0.5±1); Alanine (- 0.5); Histidine (-0.5); Cysteine (-1.0); Methionine (-1.3); Valine (-1.5); Leucine (-1.8); Isoleucine ( -1.8); Tyrosine (-2.3); Phenylalanine (-2.5); and Tryptophan (-3.4). making changes based on similar hydropathic values, including, in some embodiments, substitutions of amino acids whose hydropathic indices are within ±2, and in some embodiments, those whose hydropathic indices are within ±1, In some embodiments, those having a hydropathic index within ±0.5 are included. One can also identify epitopes from the primary amino acid sequence based on hydrophilicity. These regions are also referred to as "epitope core regions".

Table 1 shows exemplary amino acid substitutions.

Table 1: Amino Acid Substitutions

Suitable variants of the polypeptides mentioned herein can be determined by those skilled in the art using known techniques. In some embodiments, one skilled in the art can identify suitable sites on the molecule where activity can be altered without destroying activity by targeting to regions not believed to be important for activity. In some embodiments, one can identify residues and portions of molecules that are conserved among similar polypeptides. In some embodiments, conservative amino acid substitutions may be made even in regions important for biological activity or structure without destroying biological activity or adversely affecting polypeptide structure.

Additionally, one skilled in the art can review structure-function studies to identify residues in similar polypeptides that are important for activity or structure. Considering such a comparison, one can predict the importance of amino acid residues in a protein as compared to amino acid residues important for activity or structure in similar proteins. Those skilled in the art can select chemically similar amino acid substitutions for such predicted important amino acid residues.

One skilled in the art can also analyze the three-dimensional structure and amino acid sequence relative to structures in similar polypeptides. Based on such information, one skilled in the art can predict amino acid sequence alignments for the three-dimensional structure of antibodies. In some embodiments, one skilled in the art may choose not to make group changes to amino acid residues predicted to be on the surface of a protein, since such residues are involved in important interactions with other molecules. In addition, one skilled in the art can prepare experimental variants comprising single amino acid substitutions at each desired amino acid residue. These variants can then be screened using activity assays well known to those skilled in the art. Such variants can also be used to gather information about suitable variants. For example, if one finds that a change in a particular amino acid residue results in disrupted, undesirably reduced, or inappropriate activity, such change can be avoided. In other words, based on the information obtained from such routine experimentation, one skilled in the art can readily determine where further substitutions should be avoided, either alone or in combination with other mutations.

Numerous scientific publications have been published on the prediction of secondary structure. See Moult J., Curr. Op.in Biotech., 7 (4): 422-427 (1996), Chou et al., Biochemistry, 13 (2): 222-245 (1974); Chou et al., Biochemistry, 113 (2) Chou et al., Adv.Enzymol.Relat.Areas Mol.Biol., 47:45-148 (1978); Chou et al., Ann.Rev.Biochem., 47:251-276 and Chou et al. , Biophys. J., 26:367-384 (1979). In addition, computer programs are currently available to aid in the prediction of secondary structure. One approach to predicting secondary structure is based on homology simulations. For example, two polypeptides or proteins with greater than 30% sequence identity, or greater than 40% similarity, often have similar structural topologies. Recent growth in the Protein Structure Database (PDB) provides enhanced predictability of secondary structure, including the number of possible folds in a polypeptide or protein structure. See Holm et al., Nucl. Acid. Res., 27(1):244-247 (1999). It has been proposed (Brenner et al., Curr. Op. Struct. Biol., 7(3):369-376 (1997)) that there is a finite number of folds in a given polypeptide or protein, and that once the critical number of folds is determined, the structural Predictions will suitably become more precise.

Additional methods for predicting secondary structure include the "threading method" (Jones, D., Curr. Opin. Struct. Biol., 7(3):377-87 (1997); Sippl et al., Structure, 4(1): 15-19 (1996)), "Graph Analysis" (Bowie et al., Science, 253:164170 (1991); Gribskov et al., Meth. Enzym., 183:146-159 (1990); Gribskov et al., Proc. Nat Acad. Sci., 84(13):4355-4358 (1987)), and "Keys of Evolution" (see Holm, supra (1999), and Brenner, supra (1997)).

In some embodiments, antibody variants include antibody variants that have an altered number and/or type of glycosylation sites compared to the amino acid sequence of the parent polypeptide. In some embodiments, the protein variant includes a greater or lesser number of N-linked glycosylation sites than the native protein. An N-linked glycosylation site is characterized by the sequence: Asn-X-Ser or Asn-X-Thr, wherein the amino acid residue designated as X can be any amino acid residue except proline. Substitution of amino acid residues to create this sequence provides possible new sites for the addition of N-linked sugar chains. Alternatively, substitutions that remove this sequence will remove the N-linked sugar chain present. Also provided is the rearrangement of N-linked sugar chains, wherein one or more N-linked glycosylation sites (typically those that occur in nature) are removed and one or more new N-linked sites are created. Additional preferred antibody variants include cysteine variants in which one or more cysteine residues are deleted or substituted for another amino acid (eg, serine) compared to the parent amino acid sequence. Cysteine variants may be useful after the antibody has to refold into a biologically active configuration, for example after isolation of insoluble inclusion bodies. Cysteine variants generally have fewer cysteine residues than the native protein, and generally have an even number to minimize interactions with unpaired cysteines.

According to some embodiments, the amino acid substitutions are those that: (1) reduce susceptibility to proteolysis, (2) reduce susceptibility to oxidation, (3) alter binding affinity to form protein complexes, (4) ) alter binding affinity, and/or (4) confer or alter other physiochemical or functional properties on such polypeptides. According to some embodiments, one or more amino acid substitutions (in some embodiments, conservative amino acid substitutions) may be made in the naturally occurring sequence (in some embodiments, in the portion of the polypeptide outside of the domains that form intermolecular interactions). ). In some embodiments, a conservative amino acid substitution generally does not substantially alter the structural characteristics of the parent sequence (e.g., the substituting amino acid should not tend to disrupt helices present in the parent sequence, or disrupt other types of secondary structure that characterize the parent sequence ). Examples of polypeptide secondary and tertiary structures known in the art are described in Proteins, Structures and Molecular Principles (Creighton, eds., W.H. Freeman and Company, New York (1984)); Introduction to Protein Structure (C. Branden and J. Tooze, eds. , Garland Publishing, New York, N.Y. (1991)); and Thornton et al., Nature 354:105 (1991), which are incorporated herein by reference.

The term "polypeptide fragment" as used herein refers to a polypeptide having an amino-terminal and/or carboxy-terminal deletion. In some embodiments, fragments are at least 5 to 467 amino acids long. It is understood that in some embodiments, fragments are at least 5, 6, 8, 10, 14, 20, 50, 70, 100, 150, 200, 250, 300, 350, 400, or 450 amino acids in length.

Peptide analogs are commonly used in the pharmaceutical industry as non-peptide drugs with similar properties to the template peptide. These types of non-peptidic compounds are referred to as "peptidomimetics". Fauchere, J. Adv. Drug Res. 15: 29 (1986); Veber and Freidinger TINS p. 392 (1985); and Evans et al. J. Med. Chem. 30: 1229 (1987), which are hereby cited for any purpose Reference. Such compounds are often developed with the aid of computerized molecular models. Peptide mimetics that are structurally similar to therapeutically useful peptides can be used to produce similar therapeutic or prophylactic effects. In general, a peptidomimetic is structurally similar to an exemplary polypeptide (i.e., has biochemical or pharmaceutical properties), such as a human antibody, but has one optionally replaced by a bond selected from the group consisting of or multiple peptide bonds: -CH ₂ NH-, -CH ₂ S-, -CH ₂ -CH ₂ -, -CH=CH- (cis and trans), -COCH ₂ -, -CH(OH)CH ₂ -, and -CH ₂ SO-. Systematic substitution of one or more amino acids with a consensus sequence of D-amino acids of the same type (eg, D-lysine for L-lysine) can be used in some embodiments to prepare more stable peptides. In addition, constrained peptides comprising consensus sequences or substantially identical consensus sequence variants can be prepared by methods well known in the art (Rizo and Gierasch, Ann. Rev. Biochem. 61:387 (1992), cited here for any purpose for reference); for example, by adding an internal cysteine residue capable of forming an intramolecular disulfide bridge that cyclizes the peptide.

"Antibody" or "antibody peptide" refers to an intact antibody, or a binding fragment thereof that competes with the intact antibody for specific binding. In some embodiments, binding fragments are produced by recombinant DNA techniques. In some embodiments, binding fragments are prepared by enzymatic or chemical cleavage of intact antibodies. Binding fragments include, but are not limited to, Fab, Fab', F(ab')2, Fv, and single chain antibodies.

The term "heavy chain" includes any polypeptide having sufficient variable region sequence to confer specificity for OPGL. The term "light chain" includes any polypeptide having sufficient variable region sequence to confer specificity for OPGL. The full-length heavy chain includes a variable region domain, _VH , and three constant region domains, _CH1 , _CH2 , and _CH3 . The _VH domain is at the amino terminus of the polypeptide and the _CH3 domain is at the carboxy terminus. The term "heavy chain" as used herein includes full-length heavy chains and fragments thereof. A full-length light chain includes a variable region domain, V _L , and a constant region domain, _CL . Like the heavy chain, the variable region domain of the light chain is at the amino terminus of the polypeptide. The term "light chain", as used herein, includes full-length light chains and fragments thereof. The Fab fragment consists of a light chain and _CH1 and the variable regions of a heavy chain. The heavy chain of a Fab molecule cannot form a disulfide bond with another heavy chain molecule. The Fab' fragment consists of a light chain and a heavy chain, which contains more constant regions between the _CH1 and _CH2 domains, so that interchain disulfide bonds can be formed between the two heavy chains to form F (ab')2 molecules. The Fv region includes variable regions from the heavy and light chains, but no constant regions. Single-chain antibodies are Fv molecules in which the variable regions of the heavy and light chains are linked by a flexible linker to form a single-chain polypeptide, which forms the antigen-binding region. Single chain antibodies are discussed in detail in WO 88/01649 and US Patent Nos. 4,946,778 and 5,260,203.

In some embodiments, bivalent antibodies are not "multispecific" or "multifunctional" antibodies, with the general understanding that each binding site is the same.

Antibody substantially inhibits when excess antibody reduces the amount of receptor bound to the counter-receptor by at least about 20%, 40%, 60%, 80%, 85%, or more (as determined by an in vitro competition binding assay). Adhesion of ligand to receptor.

The term "epitope" includes any polypeptide determinant capable of specifically binding to an immunoglobulin or T-cell receptor. In some embodiments, epitopic determinants include chemically active surface groups of molecules such as amino acids, sugar side chains, phosphoryl groups, or sulfonyl groups, and, in some embodiments, may have specific three-dimensional structural characteristics, and/or specific charge characteristics. An epitope is the region of an antigen to which an antibody binds. In some embodiments, an antibody is said to specifically bind an antigen when it preferentially recognizes its target antigen in a complex mixture of proteins and/or macromolecules. In some embodiments, an antibody is said to specifically bind an antigen when the dissociation constant is ≤ 1 μM, in some embodiments, when the dissociation constant is ≤ 100 nM, in some embodiments, when the dissociation constant is ≤ 10 nM.

The term "agent" as used herein refers to a compound, a mixture of compounds, a biological macromolecule, or an extract prepared from a biological material.

As used herein, the term "label" or "labeled" refers to the insertion of a detectable label, for example by inserting a radiolabeled amino acid or attaching an avidin capable of being labeled (e.g., detectable by optical or colorimetric methods). Biotin moiety polypeptides containing fluorescent markers or enzymatically active streptavidin) for detection. In some embodiments, markers or markers may also be therapeutic. Various methods of labeling polypeptides and glycoproteins are known in the art and can be used. Examples of labels for polypeptides include, but are not limited to, the following group: radioisotopes or radionuclides (e.g. 3H, 14C, 15N, 35S, 90Y, 99Tc, 111In, 125I, 131I), fluorescent labels (e.g. FITC, nordamine , lanthanide phosphors), enzyme labels (e.g. horseradish peroxidase, β-galactosidase, luciferase, alkaline phosphatase), chemiluminescent reagents, biotin-based, predetermined secondary reporters Recognized polypeptide epitopes (eg, leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, epitope tags). In some embodiments, labels are attached via spacer arms of various lengths to reduce possible steric hindrance.

The term "biological sample" as used herein includes, but is not limited to, any quantity of material from a living or formerly living organism. Such living things include, but are not limited to, humans, mice, monkeys, rats, rabbits, and other animals. Such substances include, but are not limited to, blood, serum, urine, cells, tissues, organs, bone, bone marrow, lymph nodes and skin.

The term "osteopenic disease" includes, but is not limited to, osteoporosis, osteopenia, Paget's disease, osteolytic metastases, periodontitis, rheumatoid arthritis, and bone loss due to immobilization. In addition to these bone diseases, some cancers are known to increase osteoclast activity and induce bone resorption, such as breast cancer, prostate cancer and multiple myeloma. These cancers are now known to produce factors that lead to overexpression of OPGL in bone, and to increased osteoclast numbers and activity.

The term "agent or drug" as used herein refers to a compound or composition which, when properly administered to a patient, induces a desired therapeutic effect.

The term "modulator" as used herein is a compound that alters the activity or function of a molecule. For example, a modulator can cause some activity or function value of a molecule to be increased or decreased as compared to the activity or function value found in the absence of the modulator. In some embodiments, a modulator is an inhibitor that reduces at least one activity or functional value of a molecule. Some exemplary activities and functions of molecules include, but are not limited to, binding affinity, enzymatic activity, and signal transduction. Some exemplary inhibitors include, but are not limited to, proteins, peptides, antibodies, peptibodies, carbohydrates or small organic molecules, peptibodies are described eg in WO 01/83525.

As used herein, "substantially pure" means that the host species is a species that is predominantly present (eg, on a molar basis, it is more abundant in the composition than any other species). In some embodiments, a substantially purified fraction is a composition wherein the host species comprises at least about 50% (mole ratio) of all macromolecular species present. In some embodiments, a substantially pure composition contains about 80%, 85%, 90%, 95%, or more than 99% of all macromolecular species present in the composition. In some embodiments, the host species is purified to substantially homogeneity (impurity species in the composition cannot be detected by conventional detection methods), wherein the composition consists essentially of a single macromolecular species.

The term patient includes human and animal subjects.

In this application, the singular includes the plural unless specifically stated otherwise. In this application, the use of "or" means "and/or" unless stated otherwise. Furthermore, the term "comprises" as well as other forms such as "includes" and "included" are not limiting. Also, unless otherwise stated, the term "element" or "component" includes both elements and components comprising one unit and elements and components comprising more than one subunit.

Osteoprotegerin ligand (OPGL), a member of the tumor necrosis factor (TNF) family of cytokines, is involved in osteoclastogenesis. Increased osteoclast activity is associated with many osteopenic diseases, including postmenopausal osteoporosis, Paget's disease, osteolytic metastases, and rheumatoid arthritis. Therefore, decreased OPGL activity can lead to decreased osteoclast activity and can reduce the severity of osteopenic disease. According to some embodiments of the present invention, antibodies against OPGL may be used to treat osteopenic diseases, including but not limited to those mentioned above.

In some embodiments of the invention, fully human monoclonal antibodies directed against human osteoprotegerin ligand (OPGL) are provided. In some embodiments, amino acid sequences including heavy and light chain immunoglobulins, particularly sequences corresponding to variable regions, and nucleotide sequences encoding them are provided. In some embodiments, sequences corresponding to complementarity determining regions (CDRs), particularly from CDR1 to CDR3 are provided. According to some embodiments, hybridoma cell lines expressing such immunoglobulin molecules and monoclonal antibodies are also provided. In some embodiments, purified human monoclonal antibodies against human OPGL are provided.

The ability to clone and reconstitute megabase-sized human loci in artificial yeast chromosomes (YACs) and introduce them into the mouse germline is an important tool for elucidating the functional components of very large or roughly mapped loci and for making useful models of human disease A path is provided. Furthermore, the application of techniques such as replacing mouse loci with their human equivalents can provide unique insights into the expression and regulation of human gene products during development, their association with other systems, and their involvement in disease induction and progression.

An important implementation of such a strategy is the "humanization" of the mouse humoral immune system. Introduction of human immunoglobulin (Ig) loci into mice, in which endogenous Ig genes have been inactivated, provides the opportunity for mechanistic studies of programmed expression and assembly of antibodies and their role in B-cell development. Furthermore, such a strategy provides a source for the preparation of fully human monoclonal antibodies (Mabs). In some embodiments, fully human antibodies are expected to minimize the immunogenic and allergic responses of the mouse-innate or mouse-generated Mabs, thus, in some embodiments, increasing the efficacy and safety of the administered antibody . In some embodiments, fully human antibodies may be used, including repeated administration of antibodies, in the treatment of chronic and relapsing human diseases such as osteoporosis, inflammation, autoimmunity and cancer.

One can genetically engineer mouse strains deficient in antibody production with large fragments of the human Ig locus, and such mice are expected to produce human antibodies in the absence of mouse antibodies. Large human Ig fragments can retain large variable gene diversity, as well as proper regulation of antibody production and expression. By exploiting antibody diversity and selection and mouse mechanisms that lack immune tolerance to human proteins, reproducible human antibody repertoires in these mouse strains can be obtained with high affinity against all antigens of interest, including human antigens Antibody. Using hybridoma technology, antigen-specific human Mabs with the desired specificity can be prepared and selected.

In some embodiments, one can use constant regions from species other than human as well as human variable regions.

Naturally Occurring Antibody Structures

Naturally occurring antibody structural units generally include tetramers. Each such tetramer typically consists of two identical pairs of polypeptide chains, each pair having a full-length "light chain" (in some embodiments, approximately 25 kDa) and a full-length "heavy chain" (in some In embodiments, about 50-70 kDa). The amino-terminal portion of each chain generally includes a variable region of about 100 to 110 or more amino acids, which is generally responsible for antigen recognition. The carboxy-terminal portion of each chain generally defines the constant region responsible for effector function. Human light chains are generally classified as kappa and lambda light chains. Heavy chains are generally classified as mu, delta, gamma, alpha, or epsilon, and the antibody isotype is defined as IgM, IgD, IgG, IgA, and IgE, respectively. IgG has several subclasses, including but not limited to IgG1, IgG2, IgG3, and IgG4. IgM has several subclasses, including but not limited to IgM1 and IgM2. Similarly, IgA is divided into several subclasses including, but not limited to, IgAl and IgA2. In general, in full-length light and heavy chains, the variable and constant regions are joined by a "J" region of about 12 or more amino acids, and the heavy chain also includes a "D" region of about 10 or more amino acids. See, eg, Chapter 7 of Fundamental Immunology (Paul, W. Ed., Second Edition, Raven Press, N.Y. (1989)) (incorporated by reference in its entirety for all purposes). The variable region of each light/heavy chain pair generally forms the antigen binding site.

Variable regions generally have the same general structure of relatively conserved framework regions (FRs), connected by three hypervariable regions, also called complementarity determining regions or CDRs. The CDRs of each pair of two chains are generally aligned by framework regions, which can bind specific epitopes. From N-terminus to C-terminus, light and heavy chain variable regions generally comprise domains FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The arrangement of amino acids in each domain is generally defined according to the Kabat Sequences of Proeins of Immunological Interest (Nation Institutes of Health, Bethesda, Md. (1987 and 1991)), or Chothia&LeskJ.Mol.Biol.196:901-917(1987); Chothia et al., Nature 342:878-883 (1989).

Bispecific and bifunctional antibodies

Bispecific and diabodies are generally artificial hybrid antibodies with two different heavy chain/light chain pairs and two different binding sites. Bispecific antibodies can be prepared by a variety of methods including, but not limited to, fusion of hybridomas or linking of Fab' fragments. See, eg, Songsivilai & Lachmann Clin. Exp. Immunol. 79: 315-321 (1990), Kostelny et al., J. Immunol. 148: 1547-1553 (1992).

Antibody preparation

According to some embodiments, the invention includes antibodies that specifically bind OPGL. In some embodiments, antibodies can be prepared by immunization with full length OPGL, a soluble form of OPGL, or a fragment thereof. In some embodiments, antibodies of the invention may be polyclonal or monoclonal, and/or may be recombinant antibodies. In some embodiments, antibodies of the invention are prepared, eg, by immunizing transgenic animals capable of producing human antibodies (see, eg, PCT Published Application No. WO93/12227).

In some embodiments, the complementarity determining regions (CDRs) of the light and heavy chain variable regions of αOPGL-1 may be grafted onto framework regions (FRs) from the same or another species. In some embodiments, the CDRs of the light and heavy chain variable regions of αOPGL-1 can be grafted into consensus human FRs. To generate consensus human FRs, in some embodiments, FRs from several human heavy or light chain amino acid sequences are aligned to identify consensus amino acid sequences. In some embodiments, the FRs of the alpha OPGL-1 heavy or light chain are replaced with FRs from a different heavy or light chain. In some embodiments, rare amino acids in the FRs of the alpha OPGL-1 light and heavy chains are not substituted, while the remaining FR amino acids are substituted. Rare amino acids are specific amino acids at positions where they are not normally found in FRs. In some embodiments, the grafted variable region from αOPGL-1 can be used with a different constant region than the constant region of αOPGL-1. In some embodiments, the grafted variable region is part of a single chain Fv antibody. CDR grafting is described, for example, in US Patent Nos. 6,180,370, 5,693,762, 5,693,761, 5,585,089 and 5,530,101, incorporated by reference for any purpose.

According to some embodiments, antibodies of the invention are prepared by using transgenic mice that have inserted a substantial portion of the human antibody-producing genome but are rendered defective in the production of endogenous murine antibodies. Such mice are capable of producing human immunoglobulin molecules and antibodies, but are defective in the production of murine immunoglobulin molecules and antibodies. The techniques used to achieve this result are disclosed in the patents, applications and references published in this specification. In some embodiments, one may use, for example, those disclosed in PCT Published Application No. WO98/24893, which is hereby incorporated by reference for any purpose. See also Mendez et al., Nature Genetics 15:146-156 (1997), which is hereby incorporated by reference for any purpose.

According to some embodiments, fully human monoclonal antibodies specific for OPGL are prepared as follows. Immunize transgenic mice containing human immunoglobulin genes with the antigen of interest. Lymphocytes (eg, B-cells) from mice expressing the antibody are obtained. The cells thus recovered are fused with myeloid-type cell lines to produce immortalized hybridoma cell lines, and such hybridoma cell lines are screened and selected to identify hybridoma cell lines that produce antibodies specific for the antigen of interest. In some embodiments, the production of a hybridoma cell line that produces antibodies specific for OPGL is provided.

In some embodiments, the hybridoma cell lines AMG6.1, AMG6.4, AMG6.5, AMG7.1 and AMG7.2 produce antibodies of the invention. In some embodiments, the hybridoma cell lines AMG6.1, AMG6.4 and AMG6.5 produce antibodies of the invention. In some embodiments, antibodies of the invention bind OPGL with dissociation constants (Kd) of approximately 0.23 and 0.29 nM. In some embodiments of the invention, the antibody binds OPGL with a Kd of less than 0.23 nM.

In some embodiments, antibodies of the invention are of the IgG2 isotype. In some embodiments of the invention, the antibody comprises a human kappa light chain and a human IgG2 heavy chain. In some embodiments, antibodies of the invention are cloned for expression in mammalian cells. In some embodiments, the variable region of the antibody is linked to a constant region other than the constant region of the IgG2 isotype.

In some embodiments, conservative modifications to the heavy and light chains of αOPGL-1 (and corresponding modifications to the encoding nucleotides) result in OPGLs having similar functional and chemical characteristics to those of αOPGL-1. Antibody. Conversely, significant modifications of the functional and/or chemical characteristics of αOPGL-1 can be accomplished by selecting substitutions in the amino acid sequences of the heavy and light chains that differ significantly in their effect on maintaining the following properties: (a) the molecule in the region of action of the substitution The structure of the backbone, eg, as a sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the side chain volume.

For example, a "conservative amino acid substitution" may include the substitution of a non-natural amino acid residue for a natural amino acid residue such that there is little or no effect on the polarity or charge of the amino acid residue at that position. In addition, any native residue in the polypeptide can also be substituted by alanine, as has been previously described for "alanine scanning mutagenesis".

One skilled in the art can determine desired amino acid substitutions (whether conservative or non-conservative) when such substitutions are desired. In some embodiments, amino acid substitutions can be used to identify important residues of [alpha]OPGL-1, or to increase or decrease the affinity of antibodies for OPGL described herein.

In some embodiments, antibodies of the invention can be expressed in cell lines other than hybridoma cell lines. In some embodiments, sequences encoding specific antibodies can be used to transform suitable mammalian host cells. According to some embodiments, transformation can be performed by any known method for introducing a polynucleotide into a host cell, including, for example, packaging the polynucleotide in a virus (or viral vector) and transducing the host cell with the virus (or vector) or by Transfection methods are well known in the art, such as those exemplified in US Pat. Nos. 4,399,216, 4,912,040, 4,740,461 and 4,959,455 (these patent documents are hereby incorporated by reference for any purpose). In some embodiments, the transformation method used may vary depending on the host to be transformed. Methods for introducing heterologous polynucleotides into mammalian cells are well known in the art and include, but are not limited to, dextran-mediated transfection, calcium phosphate precipitation, 1,5-dimethyl-1,5-diazepine Undecamethylene polymethyl bromide-mediated transfection, protoplast fusion, electroporation, encapsulation of polynucleotides in liposomes, and microinjection of DNA directly into nuclei.

Mammalian cell lines available as hosts for expression are well known in the art and include, but are not limited to, the many immortalized cell lines available from the American Type Culture Collection (ATCC), including, but not limited to, Chinese hamster ovary (CHO) cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (eg Hep G2) and many other cell lines. In some embodiments, cell lines can be selected by determining which cell lines have high expression levels and produce antibodies with constitutive OPGL binding properties.

According to some embodiments, the antibodies of the invention can be used to detect OPGL in a biological sample. In some embodiments, this allows for the identification of cells or tissues that produce the protein. In some embodiments, antibodies that bind OPGL and block interaction with other binding compounds have therapeutic use in modulating osteoclast differentiation and bone resorption. In some embodiments, an anti-OPGL antibody can block OPGL binding to ODAR, which can lead to a block in the signal transduction cascade and loss of NF-kB-mediated transcriptional activation. Assays for measuring NF-kB-mediated transcriptional activation using, for example, luciferase reporter assays are well known to those skilled in the art.

In some embodiments, there is provided a method of treating a bone disorder comprising administering a therapeutically effective amount of an anti-OPGL antibody. In some embodiments, there is provided a method of treating a bone disorder comprising administering a therapeutically effective amount of an anti-OPGL antibody and another therapeutic agent. In some such embodiments, the additional therapeutic agent is administered in a therapeutically effective amount. In some embodiments, the bone disease is a disease characterized by net bone loss, including but not limited to osteopenia and osteolysis. In some embodiments, treatment with an anti-OPGL antibody inhibits the rate of bone resorption. Thus, in some embodiments, to compensate for subnormal bone formation, therapy may be applied to reduce the rate of bone resorption above normal, or to reduce bone resorption below normal. In some embodiments, antibodies can be assayed for binding to OPGL in the absence or presence of OPG and examined for their ability to inhibit OPGL-mediated osteoclastogenesis and/or bone resorption.

Conditions that may be treated according to some embodiments include, but are not limited to, the following groups:

Osteoporosis, including but not limited to primary osteoporosis, endocrine osteoporosis (including but not limited to hyperthyroidism, hyperparathyroidism, Cushing's syndrome, and acromegaly), bone Genetic and congenital forms of osteoporosis (including but not limited to osteogenesis imperfecta, homocystinuria, Menkes syndrome, Lay-Day syndrome), and osteoporosis due to immobilization of the extremities;

Paget's disease of bone (osteitis deformans) in adults and juveniles;

Osteomyelitis, an infectious disease of the bone that causes bone loss;

Hypercalcemia, including but not limited to hypercalcemia from solid tumors (including but not limited to breast, lung, and kidney) and hematologic malignancies (including but not limited to multiple myeloma, lymphoma, and leukemia), idiopathic hypercalcemia, and hypercalcemia associated with hyperthyroidism and renal dysfunction;

Osteopenia, including but not limited to postoperative osteopenia, steroid-administered-induced osteopenia, osteopenia associated with small and large bowel disease, and osteopenia associated with chronic liver and kidney disease;

Osteonecrosis, the death of bone cells, including but not limited to osteonecrosis associated with creative injury, osteonecrosis associated with Gaucher disease, osteonecrosis associated with sickle cell anemia, osteonecrosis associated with systemic lupus erythematosus, Osteonecrosis associated with rheumatoid arthritis, osteonecrosis associated with periodontal disease, osteonecrosis associated with osteolytic metastases, osteonecrosis associated with other conditions; and

Cartilage loss and joint erosion associated with rheumatoid arthritis.

In some embodiments, an anti-OPGL antibody may be used alone or with at least one additional therapeutic agent for the treatment of bone disease. In some embodiments, an anti-OPGL antibody is used in combination with a therapeutically effective amount of another therapeutic agent. Exemplary therapeutic agents that can be administered with an anti-OPGL antibody include, but are not limited to, bone morphogenetic factors designated BMP-1 through BMP-12, transforming growth factor-beta (TGF-beta) and members of the TGF-beta family; interleukin- 1 (IL-1) inhibitors, including but not limited to IL-1ra and its derivatives and Kineret ^™ , anakinra; TNFα inhibitors, including but not limited to soluble TNFα receptor, Enbrel ^™ , etanercept, anti-TNFα antibody, Remicade ^TM , infliximab, and D2E7 antibody; parathyroid hormone and its analogs; parathyroid hormone-related protein and its analogs; E-series prostaglandins; bisphosphonates (eg, alendronate and others); bone augmentation Minerals such as fluoride and calcium; non-steroidal anti-inflammatory drugs (NSAIDs), including but not limited to COX-2 inhibitors such as Celebrex ^TM , Celecoxib, and Vioxx ^TM , rofixixime; immunosuppressants such as Methotrexate or leflunomide, serine protease inhibitors, including but not limited to secretory leukocyte protease inhibitor (SLPI); IL-6 inhibitors (including but not limited to anti-IL-6 antibodies), IL-8 inhibition agents (including but not limited to anti-IL-8 antibodies), IL-18 inhibitors (including but not limited to IL-18 binding proteins and anti-IL-18 antibodies), interleukin-1 converting enzyme (ICE) modulators; fibroblast Growth Factors FGF-1 to FGF-10 and FGF Modulators; PAF Antagonists; Keratinocyte Growth Factor (KGF), KGF-related Molecules, and KGF Modulators; Matrix Metalloproteinase (MMP) Modulators; Nitric Oxide Synthesis Enzyme (NOS) modulators, including but not limited to, modulators of inducible NOS; glucocorticoid receptor modulators; glutamate receptor modulators; lipopolysaccharide (LPS) level modulators; Modulators and their mimetics.

In some embodiments, anti-OPGL antibodies are used with specific therapeutic agents to treat various inflammatory conditions, autoimmune conditions, or other agents associated with bone loss. In some embodiments, two, three or more agents may be administered, depending on the condition and desired level of treatment. In some embodiments, these agents are provided together by inclusion in the same formulation. In some embodiments, such agents and anti-OPGL antibodies are provided together by inclusion in the same formulation. In some embodiments, such agents may be provided together by inclusion in a therapeutic kit. In some embodiments, such agents and anti-OPGL antibodies may be provided together by inclusion in a therapeutic kit. In some embodiments, such reagents may be provided separately. In some embodiments, when administered by gene therapy, a gene encoding a protein agent and/or an anti-OPGL antibody may be included in the same vector. In some embodiments, genes encoding protein agents and/or anti-OPGL antibodies may be under the control of the same promoter region. In some embodiments, genes encoding protein agents and/or anti-OPGL antibodies may be in separate vectors.

In some embodiments, the invention relates to therapeutic regimens comprising an anti-OPGL antibody and at least one interleukin-1 (IL-1 ) inhibitor, and methods of treatment employing such therapeutic regimens. In some embodiments, the treatment regimen includes an anti-OPGL antibody and an IL-1 inhibitor and at least one additional molecule described herein. In some embodiments, the method of treatment uses an IL-1 inhibitor and/or a TNFα inhibitor in combination with an anti-OPGL antibody. In some embodiments, anti-OPGL antibodies in combination with IL-1 inhibitors and/or TNF[alpha] inhibitors can be used to treat conditions like asthma, rheumatoid arthritis, and multiple sclerosis.

Interleukin-1 (IL-1) is an anti-inflammatory cytokine. In some instances, IL-1 is a mediator in many diseases and medical conditions. In some instances, macrophages/monocyte lineage cells produce IL-1. In some instances, IL-1 is produced in two forms: IL-1α (IL-1α) and IL-1β (IL-1β).

This is considered if a spontaneous or experimental disease or medical condition is associated with elevated levels of IL-1 in body fluids or tissues and/or if cells or tissues from the body produce elevated levels of IL-1 in culture The disease or medical condition is an "interleukin-1 mediated disease". In some embodiments, such interleukin-1-mediated diseases are identified by the following two additional conditions: (1) the disease or medical condition can be experimentally modeled in animals by administering IL-1 or upregulating the expression of IL-1 associated pathological findings; and (2) inhibition or elimination of pathology induced in experimental animal models of the disease or medical condition by treatment with an agent that inhibits the action of IL-1. In some embodiments, one or more of the above conditions are consistent with an IL-1-mediated disease. In some embodiments, all three conditions are consistent with IL-1-mediated diseases.

Acute and chronic interleukin-1 (IL-1 )-mediated diseases include, but are not limited to, the following groups: acute pancreatitis; amyotrophic lateral sclerosis (ALS, or Lou Gehrig's disease); Alzheimer's disease; cachexia/anorexia , including but not limited to AIDS-induced cachexia; asthma and other lung diseases; atherosclerosis; autoimmune vasculitis; chronic fatigue syndrome, Clostridium-associated diseases, including but not limited to Clostridium-associated diarrhea , coronary artery conditions and indications, including but not limited to congestive heart failure, coronary restenosis, myocardial infarction, myocardial dysfunction (eg, associated with sepsis), and coronary artery bypass graft; cancer, including but not limited to Leukemia, including but not limited to multiple myeloma leukemia and myeloid leukemia (such as AML and CML), and tumor metastasis; diabetes mellitus (including but not limited to insulin-dependent diabetes mellitus); endometriosis; fever; fibromyoma; Glomerulonephritis; graft-versus-host disease and/or graft rejection; hemorrhagic shock; hyperalgesia; inflammatory bowel disease; joint inflammatory conditions, including but not limited to osteoarthritis, psoriatic arthritis, and rheumatoid arthritis inflammation; inflammatory eye diseases, including but not limited to, for example, those associated with corneal transplantation; ischemia, including but not limited to cerebral ischemia (including but not limited to brain injury as a result of, for example, trauma, epilepsy, hemorrhage or stroke, Each of these conditions can lead to neurodegeneration); Kawasaki's disease; learning disabilities; pulmonary disease (including but not limited to acute respiratory distress syndrome, or ARDS); multiple sclerosis; Muscle protein metabolism in sepsis); neurotoxicity (including but not limited to HIV-induced such conditions); osteoporosis; pain, including but not limited to pain associated with cancer; Parkinson's disease; periodontal disease; premature birth; psoriasis; reperfusion injury; septic shock; side effects of radiation therapy; temporomandibular joint disease; sleep disturbance; uveitis; Inflammation caused by the process.

In some embodiments, an IL-1 inhibitor can be any protein or molecule that specifically prevents activation of a cellular receptor to IL-1. Illustrative mechanisms include, but are not limited to, downregulation of IL-1 production, binding of free IL-1, interference with IL-1 binding to its receptor, and interference with IL-1 receptor complex formation (i.e., IL-1 receptor and IL-1 receptor accessory protein association), which interferes with the regulation of IL-1 signaling following binding to its receptor.

Some interleukin-1 inhibitors include, but are not limited to, IL-1 receptor antagonists, including but not limited to Kineret™, anakinra, IL-1ra, IL-1ra variants, and IL-1ra derivatives, collectively referred to herein as "IL-1ra "Proteins"; anti-IL-1 receptor monoclonal antibodies (see, e.g., EP623674, incorporated herein by reference for any purpose); IL-1 binding proteins, including but not limited to, soluble IL-1 receptors (see, For example, U.S. Patent No. 5,492,888, U.S. Patent No. 5,488,032, and U.S. Patent No. 5,464,937, U.S. Patent No. 5,319,071, and U.S. Patent No. 5,180,812, which are hereby incorporated by reference for any purpose); anti-IL-1 monoclonal Cloned antibodies (see, e.g., WO9501997, WO9402627, WO9006371, U.S. Patent No. 4935343, EP364778, EP267611 and EP220063, incorporated herein by reference for any purpose); IL-1 receptor accessory proteins and antibodies thereto (see, e.g., WO96/23067 and WO99/37773, incorporated herein by reference for any purpose); inhibitors of interleukin-1β converting enzyme (ICE) or caspase (see, e.g., WO99/46248, WO99/47545, and WO99/47154, for incorporated herein by reference for any purpose), which can be used to inhibit IL-1β production and secretion; interleukin-1β protease inhibitors; and other compounds and proteins that block the in vivo synthesis or extracellular release of IL-1.

Exemplary IL-1 inhibitors are disclosed in, for example, US Patent Nos. 5,747,444, 5,359,032; 5,608,035; 5,843,905; 5,359,032; , 91/17184, 96/40907, 98/32733, 98/42325, 98/44940, 98/47892, 98/56377, 99/03837, 99/06426, 99/06042, 91/17249, 98/32733, 98 /17661, 97/08174, 95/34326, 99/36426, 99/36415; European patent applications (EP) 534978 and 894795; and French patent application FR2762514. The disclosures of all of the above references are hereby incorporated by reference for any purpose.

Interleukin-1 receptor antagonist (IL-1ra) is a human protein that acts as a natural inhibitor of interleukin-1 and is a member of the IL-1 family, including IL-1α and IL-1β. Some receptor antagonists, including IL-1ra and variants and derivatives thereof, and methods of making and using them are described in US Patent Nos. 5075222; WO91/08285; WO91/17184; AU9173636; WO92/16221; WO93/21946 WO94/06457; WO94/21275; FR2706772; WO94/21235; DE4219626; WO94/20517; WO96/22793; WO97/28828; In some embodiments, IL-1 receptor antagonists may be glycosylated. In some embodiments, IL-1 receptor antagonists may be aglycosylated.

Three forms of IL-1ra and variants thereof are described in US Patent No. 5,075,222 (the '222 patent). The first form is called "IL-1i" in U.S. Patent No. 5,075,222 and is characterized as a 22-23kD molecule on SDS-PAGE with an isoelectric point of about 4.8 obtained from a Mono QFPLC column containing about 52mM NaCl Tris buffer, pH 7.6 for elution. The second form, IL-1raβ, was characterized as a 22-23kD protein, eluted from a Mono Q column with 48mM NaCl. IL-1raα and IL-1raβ are glycosylated. The third form, IL-1rax, is characterized as a 20 kD protein, eluted from a Mono Q column with 48 mM NaCl, and is non-glycosylated. U.S. Patent No. 5,075,222 also describes methods for isolating genes encoding inhibitors, cloning those genes in suitable vectors, transforming and transfecting those genes into certain cell types, and expressing those genes to produce inhibitory agent method.

In some embodiments, deletions, insertions, and/or substitutions (individually or collectively "variants") are made in the IL-1ra amino acid sequence. In some embodiments, the IL-1ra variant is biologically active (eg, has the ability to inhibit IL-1).

In some embodiments, the invention relates to a treatment regimen comprising an anti-OPGL antibody and at least one TNFα inhibitor, and methods of using the same. In some embodiments, the treatment regimen includes an anti-OPGL antibody and a TNFα inhibitor and at least one additional molecule described herein.

Several diseases and medical conditions are mediated by TNF and are classified as inflammatory conditions. "TNF-mediated disease" as used herein includes, but is not limited to, diseases associated with elevated levels of TNF in body fluids or tissues and/or in which cells or tissues are obtained from individuals producing high levels of TNF in culture or medical condition. In some embodiments, if (1) pathological findings associated with a disease or medical condition can be experimentally mimicked in animals by administering or upregulating TNF expression and/or (2) can be inhibited or destroyed by treatment with an agent that inhibits the action of TNF A disease is considered to be a TNF-mediated disease if pathology is induced in an experimental animal model of the disease or medical condition.

Several diseases and medical conditions are TNF-mediated and can be classified as inflammatory conditions. As used herein, "TNF-mediated disease" includes, but is not limited to: cachexia and anorexia; cancer, including, but not limited to, leukemia; chronic fatigue syndrome; coronary artery conditions and/or indications, including, but not limited to, hyperemia heart failure, coronary restenosis, myocardial infarction, myocardial dysfunction (including but not limited to such conditions associated with sepsis), and coronary artery bypass grafting; depression; diabetes mellitus, including but not limited to, juvenile-onset Type 1 diabetes, diabetes, and insulin resistance (including but not limited to insulin resistance associated with obesity); endometriosis, endometritis, and related conditions; fibromyoma and analgesia; graft-resistant Host rejection; hyperalgesia; inflammatory bowel disease, including but not limited to Crohn's disease and Clostridium difficile-associated diarrhea; ischemia, including but not limited to, brain injury as a result of trauma, seizures, hemorrhage, and/or stroke Pulmonary diseases, including but not limited to adult respiratory distress syndrome, asthma, pulmonary fibrosis; Multiple sclerosis; Neuroinflammatory diseases; Including, but not limited to, cancer-related pain; pancreatitis, periodontal disease; pityriasis pilaris (PRP); prostatitis, including bacterial and nonbacterial prostatitis, and related conditions; psoriasis and related conditions; pulmonary fibrosis Reperfusion injury; Rheumatism, including but not limited to rheumatoid arthritis, osteoarthritis, juvenile arthritis (including but not limited to juvenile rheumatoid arthritis), seronegative polyarthritis, ankylosing spondylitis , Reiter's syndrome and reactive arthritis, Still's disease, psoriatic arthritis, enteropathic arthritis, polymyositis, dermatomyositis, scleroderma, systemic sclerosis, vasculitis (e.g. Kawasaki's disease), cerebrovascular arthritis, Lyme disease, staphylococcal-induced ("septic") arthritis, Sjogren's syndrome, rheumatic fever, polychondritis and polymyopathy rheumatica, and giant cell arteritis); septic shock; radiotherapy Side effects; systemic lupus erythematosus (SLE); temporomandibular joint disease; thyroiditis; and tissue transplantation and/or, for example, caused by strain, sprain, cartilage damage, trauma, orthopedic surgery, infection (eg, HIV, Clostridium difficile and related species) or inflammation caused by other disease processes.

In some embodiments, the TNF inhibitor acts by at least one of downregulating or inhibiting TNF production, binding free THF, interfering with binding of TNF to its receptor, and interfering with modulation of TNF signaling following binding to its receptor. The term "TNF inhibitor" includes, but is not limited to, soluble TNF receptors, including, but not limited to, soluble tumor necrosis factor receptor type I (sTNF-RI; also known as p55 receptor), soluble tumor necrosis factor receptor type II ( also known as p75 receptor), and Enbrel ^™ , etanercept; anti-TNF antibodies including, but not limited to, Remicade ^™ , infliximab and D2E7 (see, eg, US Patent Nos. 6,090,382 and 6,258,562); anti-TNF receptor antibodies; sTNF-RI (see, eg, WO98/24463), Etanercept (Enbrel ^™ ); inhibitors of TNF-alpha converting enzyme (TACE); and other molecules that affect TNF activity.

Exemplary TNF-alpha inhibitors are described, for example, in European Patent Application

EP308378；EP422339；EP393438；EP398327；EP412486；EP418014，EP417563，EP433900；EP464533；EP512528；EP526905；EP568928；EP607776，其中描述了来氨米特抑制TNF-α的用途；EP663210；EP542795；EP818439；EP664128；EP542795； EP741707；EP874819；EP882714；EP880970；EP648783；EP731791；EP895988；EP550376；EP882714；EP853083；EP550376；EP943616；EP939121；EP614984；EP853083；美国专利号5,136,021；5,929,117；5,948,638；5,807,862；5,695,953；5,834,435；5,817,822；5830742；5,834,435 ；5,851,556；5,853,977；5,359,037；5,512,544；5,695,953；5,811,261；5,633,145；5,863,926；5,866,616；5,641,673；5,869,677；5,869,511；5,872,146；5,854,003；5,856,161；5,877,222；5,877,200；5,877,151；5,886,010；5,869,660；5,859,207；5,891,883；5,877,180；5,955,480；5,955,476 5,955,435; 5,994,351; 5,990,119; 5,952,320; WO96/28546, WO98/27298, WO98/30541, WO96/38150, WO96/38150, WO97/18207, WO97/15561, WO97/12902, WO96/25861, WO96/12735, WO96/11209, WO98/39326, WO98/ 39316, WO98/38859, WO98/39315, WO98/42659, WO98/3932 9. WO98/43959, WO98/45268, WO98/47863, WO96/33172, WO96/20926, WO97/37974, WO97/37973, WO97/47599, WO96/35711, WO98/51665, WO98/43946, WO95/04045, WO98/56377, WO97/12244, WO99/00364, WO99/00363, WO98/57936, WO99/01449, WO99/01139, WO98/56788, WO98/56756, WO98/53842, WO98/52948, WO98/52937, WO99/ 02510, WO97/43250, WO99/06410, WO99/06042, WO99/09022, WO99/08688, WO99/07679, WO99/09965, WO99/07704, WO99/06041, WO99/37818, WO99/37625, WO97/11668, WO99/50238, WO99/47672, WO99/48491; Japanese patent applications 10147531, 10231285, 10259140, and 10130149, 10316570, 11001481, and 127,800/1991; German patent applications 19731525;

The disclosures of all above-mentioned references are hereby incorporated by reference for any purpose.

EP393438 and EP422339 describe soluble TNF receptor type I (also known as sTNFR-I or 30kDa TNF inhibitor) and soluble TNF receptor type II (also known as sTNFR-II or 40kDa TNF inhibitor), collectively referred to herein as "sTNFRs". EP393438 and EP422339 also describe modified forms of sTNFR-I and sTNFR-II, including but not limited to, fragments, functional derivatives, and variants. Furthermore, EP393438 and EP422339 describe methods for isolating genes encoding inhibitors, cloning genes in suitable vectors, transforming and transfecting genes into certain cell types, and expressing genes to produce inhibitors.

sTNFR-I and sTNFR-II are members of the receptor superfamily of nerve growth factor/TNF receptors, including nerve growth factor receptor (NGF), B cell antigen CD40, 4-1BB, rat T-cell antigen MR COX40, The fas antigen, and the CD27 and CD30 antigens (Smith et al. (1990) Science 248: 1019-1023). A conserved feature of this group of cell surface receptors is the cysteine-rich extracellular ligand-binding domain, which can be divided into groups of approximately 40 amino acids containing 4–6 cysteine residues at very conserved positions. Four repeat motifs (Smith et al. (1990), supra).

EP393438 discloses the 40kDa TNF inhibitor Δ51 and the 40kDa TNF inhibitor Δ53, which are truncated forms of the full length recombinant 40kDa TNF inhibitor protein. Δ51 and Δ53 have deletions of 51 or 53 amino acids, respectively, at the carboxy-terminus of the mature protein.

Published PCT Application No. WO98/01555 describes a domain that does not contain a fourth domain (amino acid residues Thr ¹²⁷ -Asn ¹⁶¹ of sTNFR-I and amino acid residues Pro ¹⁴¹ -Thr ¹⁷⁹ of sTNFR-II); A portion (amino acid residues Asn ¹¹¹ -Cys ¹²⁶ of sTNFR-I and amino acid residues Pro ¹²³ -Lys ¹⁴⁰ of sTNFR-II); and optionally, a portion that does not contain the first domain (amino acid residues of sTNFR-I Asp ¹ -Cys ¹⁹ and amino acid residues Leu ¹ -Cys ³² of sTNFR-II) truncated forms of sTNFR-I and sTNFR-II. In some embodiments, truncated sTNFRs include proteins represented by the formulas R ₁ -[Cys ¹⁹ -Cys ¹⁰³ ]-R ₂ and R ₄ -[Cys ³² -Cys ¹¹⁵ ]-R ₅ . These proteins are truncated forms of sTNFR-I and sTNFR-II, respectively.

As used herein, "R ₁ -[Cys ¹⁹ -Cys ¹⁰³ ]-R ₂ " represents one or more proteins, wherein [Cys ¹⁹ -Cys ¹⁰³ ] are residues 19 to 103 of sTNFR-1, WO98/01555 Its sequence is provided in Figure 1; wherein R ₁ represents the methionated or non-methionated amine group of Cys ¹⁹ or one or more amino-terminal amino acid residues selected from Cys ¹⁸ to Asp ¹ ; and wherein R ₂ represents the carboxyl group of Cys ¹⁰³ or one or more carboxy-terminal amino acid residues selected from Phe ¹⁰⁴ to Leu ¹¹⁰ .

Exemplary truncated sTNFR-I of the present invention include but not limited to sTNFR-I2.6D/C105, sTNFR-I2.6D/C106, sTNFR-I2.6D/N105, sTNFR-I2.3D/d8, sTNFR-I2. 3D/d18, sTNFR-I2.3D/d15, either methylated or non-methylated, variants and derivatives thereof. Some exemplary truncated sTNFR-I are described, eg, in Published PCT Application No. WO98/01555.

As used herein, "R ₃ -[Cys ³² -Cys ^11} -R ₄ " represents one or more proteins, wherein [Cys ³² -Cys ¹¹⁵ ] are residues Cys ³² to Cys ¹¹⁵ of sTNFR-II, WO98 Figure 8 of /01555 provides its sequence; wherein _R represents the methionated or non-methionated amine group of Cys ³² or one or more amino-terminal amino acid residues selected from Cys ³¹ to Leu ¹ and wherein R ₄ represents the carboxyl group of Cys ¹¹⁵ or one or more carboxy-terminal amino acid residues selected from A 1 a ¹¹⁶ to Ar g ¹²² .

In some embodiments, the invention relates to therapeutic regimens comprising an anti-OPGL antibody and at least one serine protease inhibitor, and methods of treatment employing such therapeutic regimens. In some embodiments, the treatment regimen includes an anti-OPGL antibody and a serine protease inhibitor and at least one additional molecule described herein.

Endogenous proteolytic enzymes can degrade invading organisms, antigen-antibody complexes, and some tissue proteins that are no longer needed or useful. Infectious substances can introduce additional proteolytic enzymes into the organism. Protease inhibitors can regulate endogenous and invading proteolytic enzymes.

In some embodiments, naturally occurring protease inhibitors control endogenous proteases by locally and temporarily limiting their response. In some embodiments, protease inhibitors inhibit proteases introduced into the body by infectious agents. In some instances, tissues that are particularly susceptible to proteolytic attack and infection, including but not limited to those of the respiratory tract, are enriched in protease inhibitors.

Protease inhibitors make up approximately 10% of human plasma proteins. At least eight inhibitors have been isolated from this source and characterized in the literature. These include, but are not limited to, α2-macroglobulin (α2M), α1-protease inhibitor (α1PI), α1-antichymotrypsin (α1Achy), α1-anticollagenase (α1AC), and endo-α-pancreatic Protease Inhibitor (I-alpha-I).

In some cases, disruption of the protease/protease inhibitor balance can lead to protease-mediated tissue destruction, including but not limited to, emphysema, arthritis, glomerulonephritis, periodontitis, muscular atrophy, tumor invasion, and various other pathological conditions. In some cases, such as severe pathological processes like sepsis or acute leukemia, the amount of free proteolytic enzymes present is increased due to the release of the enzymes from secreting cells.

Furthermore, in some cases, reducing the ability of an organism to regulate inhibitors can also lead to changes in the protease/protease inhibitor balance. A non-limiting example of such reduced ability to regulate inhibitors is alpha 1 -protease inhibitor deficiency, which is associated with emphysema development.

In some cases, severe damage to organisms can occur when such abnormal conditions exist unless measures can be taken to control proteolytic enzymes. Accordingly, protease inhibitors that can be administered to organisms to control proteolytic enzymes have been sought.

Leukocyte elastase, trypsin, cathepsin G, and pancreatic elastase are non-limiting examples of the class of proteases known as serine proteases.

In some cases, when released extracellularly, leukocyte elastase degrades connective tissue and other valuable proteins. Although normally functioning organisms degrade certain amounts of connective tissue and other proteins, the presence of excess leukocyte elastase may be associated with various pathological conditions including, but not limited to, emphysema and rheumatoid arthritis. In some embodiments, to block the action of leukocyte elastase, a protease inhibitor specific for leukocyte elastase is sought when it is present in greater than normal amounts. Such a protease inhibitor is useful if it can be isolated or prepared in a pharmaceutically useful pure form and in sufficient quantity.

Some leukocyte elastase inhibitors are described, for example, in Schiessler et al., "Acid-Stable Inhibitors of Giant Cell Neutral Proteases in Human Mucus Secretion," Neutral Proteases of Human Polymorpheuclear Leucocytes, Havemann et al. (eds.), Urban and Schwarzenberg, Inc. (1978), and Travis and Salvesen, Ann. Rev. Biochem. 52:655-709 (1983).

In some instances, trypsin causes the degradation of some soft organ tissues, such as pancreatic tissue, during a variety of acute conditions, including but not limited to pancreatitis. Trypsin inhibitors are useful if they can be isolated or prepared in a pharmaceutically useful pure form and in sufficient quantities.

Cathepsin G is another protease present in white blood cells. In some embodiments, cathepsin G is capable of degrading various proteins in vitro, including those of the complement pathway. Pancreatic elastin is another protease that plays a role in pancreatitis. Therefore, inhibitors of these proteases may also be of medicinal value.

In some embodiments, it is believed that substrate specificity and sensitivity to different inhibitors of serine proteases is the result of changes in only a few amino acid residues. Similarly, it is possible to conceive a class of serine protease inhibitors in which relatively few amino acid changes result in the inhibition of different proteases. In some embodiments, members of this class of inhibitors inhibit each serine protease.

Exemplary serine protease inhibitors are secretory leukocyte protease inhibitor (SLPI) and fragments and analogs thereof. Exemplary serine protease inhibitors also include, but are not limited to, anti-leukocyte protease (ALP), mucus protease inhibitor (MPI), human semen plasma inhibitor-1 (HUSI-1), bronchial mucus inhibitor (BMI), and Cervical Mucus Inhibitor (CUSI). In some embodiments, a serine protease inhibitor may also be an LPS modulator. See, eg, Jin et al. (1997), Cell 88(3):417-26. In some embodiments, these molecules are suitable for use in conditions that lead to bone loss because they preferentially involve cartilage.

Exemplary serine protease inhibitors are described, for example, in US Patent No. 4,760,130; US Patent No. 5,900,400; and US Patent No. 5,633,227; these patent documents are hereby incorporated by reference for any purpose. The molecules disclosed in the above documents and all their variants or analogs are collectively referred to as "serine protease inhibitors".

IL-18 is a pro-inflammatory cytokine that was found to induce interferon-gamma and was formerly known as interferon gamma-inducible factor (IGIF). In some cases, IL-1 has been shown to upregulate IL-18 production, and IL-18 induces the production of a number of pro-inflammatory cytokines, including IL-6 and MMP-1. See, eg, Dinarello et al. (1998), J. Leukocyte Biol. 63:658-64. In some cases, caspase I is also important for IL-18 production. Experiments also suggest that TNF-α regulates the production of IL-18, and simultaneously inhibiting TNF-α and IL-18 protects the liver from toxicity. See, eg, Faggioni et al. (2000), PNAS 97:2367-72.

IL-18 acts in vivo by recalling the receptor system of the IL-1 system. IL-18 interacts with a cell surface receptor (IL-18R), which interacts with an accessory protein (IL-18RAcP). IL-18-mediated signaling occurs when the IL-18, IL-18R and IL-18RacP complex is formed. A natural inhibitor of IL-18 is IL-18bp. In some embodiments, IL-18bp functions as a "decoy receptor" by binding IL-18 molecules and preventing interaction with IL-18R.

In some embodiments, the invention relates to therapeutic regimens comprising an anti-OPGL antibody and at least one IL-18 inhibitor, and methods of treatment employing such therapeutic regimens. In some embodiments, the treatment regimen includes an anti-OPGL antibody and an IL-18 inhibitor and at least one additional molecule described herein. Exemplary conditions that may be treated according to some embodiments include, but are not limited to, inflammation, autoimmune disease, IL-1 mediated disease, and TNF-mediated disease. Exemplary conditions that may be treated with an anti-OPGL antibody and at least one IL-18 inhibitor according to some embodiments include, but are not limited to, arthritis, including but not limited to rheumatoid arthritis; systemic lupus erythematosus (SLE); transplants Anti-host disease (GvHD); hepatitis, sepsis; and bone and cartilage loss associated with these diseases.

Exemplary IL-18 inhibitors include, but are not limited to, antibodies that bind IL-18; antibodies that bind IL-18R; IL-18RAcP; IL-18bp; IL-18R fragments (e.g., soluble IL-18 receptor Antibodies that bind IL-18 and reduce or prevent its interaction with IL-18R; bind IL-18R and reduce or prevent its interaction with IL-18 or with IL-18RAcP Peptides that bind to IL-18RAcP and reduce or prevent its interaction with IL-18R; peptides that reduce or prevent IL-18 production or the interaction between IL-18, IL-18R and IL-18RAcP molecular.

Some IL-18 inhibitors are described, for example, in US Patent No. 5912324, issued Jul. 14, 1994; EP0962531, published Dec. 8, 1999; EP712931, published Nov. 15, 1994; Jul. 14, 1994 U.S. Patent No.5914253 issued on July 10, 1997; U.S. Patent No.6060283 issued on May 9, 2000; EP850952 published on December 26, 1996; September 16, 1998 EP864585 published in Japan; WO98/41232 published on September 24, 1998; U.S. Patent No.6054487 issued on April 25, 2000; WO99/09063 published on August 14, 1997; published on November 3, 1997 WO99/22760 published on January 23, 1998; WO99/37773 published on March 20, 1998; EP0974600 published on January 26, 2000; WO00/12555 published on March 9, 2000 ; Japanese patent application JP111399/94 published on October 31, 1997; Israeli patent application IL121554AO published on February 8, 1998; these patent documents are hereby incorporated by reference for any purpose.

In some embodiments, an anti-OPGL antibody can be administered with at least one therapeutic agent for inflammation. In some embodiments, an anti-OPGL antibody can be administered with at least one therapeutic agent for an immune disease. Exemplary therapeutic agents for inflammatory and immune disorders include, but are not limited to, corticosteroids, including, but not limited to, prednisolone; non-steroidal anti-inflammatory drugs (NSAIDs), including, but not limited to, cyclooxygenase type 1 ( COX-1) and cyclooxygenase type 2 (COX-2) inhibitors; disease modifying antirheumatic drugs (DMARDs), including but not limited to, methotrexate, hydroxychloroquine, chloroquine, cyclosporine, Gold compounds (e.g., glucoraphanate, gold thiosuccinate, and glucothiogold), leflunomide; type IV phosphodiesterase inhibitors including, but not limited to, rolipram and hexanone cocoa base; tacrolimus (FK-506); sirolimus (rapamycin); mycophenolic acid; 5-lipoxygenase inhibitors, including but not limited to, zileuton; interleukin-6 (IL- 6) Modulators; small molecule modulators of 38kDa mitogen-activated protein kinase (p38-MAPK); small molecule modulators of intracellular molecules involved in inflammatory pathways, wherein such intracellular molecules include, but are not limited to, jnk , IKK, NF-Kb, ZAP70, and lck. Some exemplary therapeutic agents for inflammation are described, eg, in CADinarello and LL Moldawer Proinflammatory and Anti-Inflammatory Cytokines in Rheumatoid Arthritis: A Primer for Clinicians Third Edition (2001) Amgen Inc. Thousand Oaks, CA. Exemplary therapeutic agents for inflammatory and autoimmune diseases include, but are not limited to, interferon-gamma (IFN-γ) modulators; modulators of OX40/OX40L (including OX40 in soluble form); 4-1BB/4 - modulators of 1BB ligands (including soluble forms of 4-1BB); and modulators of the B cell-T cell co-stimulatory pathway, including but not limited to, receptor ligand pairs CD28/B7, CD40/CD40L, ICOS /B7RP1, and modulator of AGP-3/TACI/BAFFR (AGP-3 binds both TACI and BAFFR receptors). Some exemplary regulators of the B cell-T cell co-stimulatory pathway include, but are not limited to, CD28, B7.1 and B7.2 (including soluble forms of B7.1 or B7.2 and soluble forms of CTLA4, which The two can be fused into a heterologous peptide or protein that reduces or prevents degradation and/or increases half-life, reduces toxicity, reduces immunogenicity, or increases the biological activity of a therapeutic protein by increasing solubility or circulating half-life); CD40 and CD40L Inhibitors (including soluble forms of CD40 that can be fused to heterologous peptides or proteins); inhibitors of ICOS and B7RP1 (including soluble forms of ICOS that can be fused to heterologous peptides or proteins) and AGP-3, TACI and Inhibitors of BAFFR (including soluble forms of TACI and BAFFR). ICOS, B7RP1 and inhibitors thereof are described, eg, in WO00/46240. AGP-3, TACI and BAFFR and inhibitors thereof are described, eg, in WO00/47740, WO01/85872, WO02/15273, WO98/39361, and Bulow and Bram (1997) Science 278:138-140.

In some embodiments, anti-OPGL antibodies are used to treat bone loss including, but not limited to, bone loss resulting from osteolytic destruction of bone caused by malignant or metastatic tumors. In some embodiments, anti-OPGL antibodies can be used to treat bone loss associated with cancer. Exemplary cancers include, but are not limited to, breast, prostate, thyroid, kidney, lung, esophagus, rectum, bladder, cervix, ovary, and liver, and gastrointestinal tract. In some embodiments, anti-OPGL antibodies can be used to treat bone loss associated with, for example, some hematological malignancies, including, but not limited to, multiple myeloma and lymphoma, including Hodgkin's disease.

In some embodiments, the anti-OPGL antibody is administered alone. In some embodiments, an anti-OPGL antibody is administered with at least one other therapeutic agent, including, but not limited to, at least one other cancer therapeutic agent. Exemplary cancer therapeutics include, but are not limited to, radiation therapy and chemotherapy. In some embodiments, chemotherapy may include treatment with one or more of the following drugs: anthracycline, paclitaxel, tamoxifen, doxorubicin, 5-fluorouracil, and others known in the art. In some embodiments, the cancer therapeutic agent is a luteinizing hormone releasing hormone (LHRH) antagonist. In some embodiments, the LHRH antagonist is a peptide antagonist.

In some embodiments, the LHRH antagonist comprises the peptide: Ac-D-Nal-4-Cl-Phe-D-Pal-Ser-N-Me-Tyr-D-Asn-Leu-Lys(iPr)-Pro-D -Ala-NH2 (SEQ ID NO: 20), wherein Nal is 3-(2-naphthyl)alanyl; 4-Cl-Phe is (4'-chlorophenyl)alanyl; Pal is 3-( 3'-pyridyl)alanyl; and Lys(iPr) is N-ε-2-propyl-lysyl.

In some embodiments, the LHRH antagonist is an LHRH antagonist decapeptide. Some exemplary decapeptides are described, for example, in US Patent No. 5,843,901, which is hereby incorporated by reference for any purpose.

Exemplary therapeutic antibodies according to some embodiments include, but are not limited to, mouse, mouse-human chimeric, CDR-grafted, humanized and fully human antibodies, and synthetic antibodies, including, but not limited to, those selected by screening antibody libraries Those ones. Exemplary antibodies include, but are not limited to, binding to the cell surface proteins Her2, CDC20, CDC33, mucin-like glycoproteins, and epidermal growth factor receptor (EGFR) present on tumor cells, and optionally inducing response to the expression of these proteins. Cytostatic and/or cytotoxic effects on tumor cells. Exemplary antibodies also include HERCEPTIN ^™ (ibritumomab tiuxetan), which may be used to treat breast cancer and other forms of cancer, and RITUXAN ^™ (rituximab), ZEVALIN ^™ (ibritumomab tiuxetan), and LYMPHOCIDE ^™ (epratuzumab) , which can be used to treat non-Hodgkin's lymphoma and other forms of cancer. Some exemplary antibodies also include ERBITUX ^™ (IMC-C225), BEXXAR ^™ (I ¹³¹ tositumomab), and Campath.

In some embodiments, the cancer therapeutic agent is a polypeptide that selectively induces apoptosis in tumor cells, including, but not limited to, the TNF-related polypeptide TRAIL. In some embodiments, the anti-OPGL antibody is administered simultaneously and possibly sequentially prior to treatment with at least one cancer therapeutic. In some embodiments, anti-OPGL antibodies may be administered prophylactically in order to prevent or reduce bone loss from metastatic cancer. In some embodiments, an anti-OPGL antibody can be administered to treat a condition of bone loss due to metastasis.

In some embodiments, anti-OPGL antibodies can be used to prevent and/or treat bone loss associated with multiple myeloma and/or prevent and/or treat the disease itself. Multiple myeloma is a tumor of B cell origin that can cause significant morbidity and/or mortality. In some embodiments, multiple myeloma is clinically manifested by focal bone loss, possibly due to localized areas of increased osteoclast activation. Many myeloma patients have bone lesions and bone pain by radiological analysis. In some cases, myeloma patients are prone to pathologic fractures, which may occur spontaneously or due to injury. In some cases, the skeletal lesions that develop during myeloma result not just in fractures but also in deformities and, occasionally, nerve compression, especially in the spinous processes. In some patients, pathological increases in serum calcium (hypercalcemia) occur, causing significant problems in the treatment of the disease. In some embodiments, anti-OPGL antibodies can be administered to patients to reduce or block bone resorption and calcium release, which can reduce the risk of fractures and spinal deformities.

In some cases, myeloma cells do not directly participate in bone destruction, but instead produce extracellular signals that lead to osteoclast differentiation and activation. In some cases, osteoclasts produce high levels of the cytokine IL-6, especially when they become activated. IL-6 is a B cell growth factor and is responsible for the growth of murine and human myeloma cells in vitro. Myeloma cells also directly or indirectly produce OPGL, which can cause localized osteolysis around myeloma cells embedded in the bone marrow cavity. In some cases, normal osteoclasts in close proximity to myeloma cells then produce IL-6, which can lead to local expansion of tumor cells. Myeloma cells expand clonally and occupy the bone cavity that results from inappropriate bone resorption.

Administration of OPG to rodents has been observed to induce rapid death of osteoclast populations (see, eg, Lacey et al. (2000) Am. J. Pathol. 157:435-448). A reduction in the number of osteoclasts hinders the contribution of those cells to increased IL-6 production, and thus affects the growth and survival of myeloma cells in trabecular bone. Thus, in some embodiments, administration of anti-OPGL antibodies to myeloma patients not only blocks hyper resorption of bone, but also affects the expansion and survival of the tumor itself.

B-cells express the receptor for OPGL, ODAR. Myeloma cells also express ODAR and additionally produce OPGL. In some cases, expression of both OPGL and ODAR in the same cell population can produce autocrine stimuli that affect myeloma cell survival. Thus, in some embodiments, administration of anti-OPGL antibodies can reduce tumor cell survival, thereby reducing or eliminating tumor burden in myeloma patients.

In some embodiments, the present invention provides a pharmaceutical composition comprising a therapeutically effective amount of an anti-OPGL antibody and a pharmaceutically acceptable diluent, carrier, solubilizer, emulsifier, preservative and/or adjuvant.

In some embodiments, the present invention provides an anti-OPGL antibody comprising a therapeutically effective amount and a therapeutically effective amount of at least one additional therapeutic agent, and a pharmaceutically acceptable diluent, carrier, solubilizer, emulsifier, preservative and/or Adjuvant pharmaceutical composition. In some embodiments, the at least one additional therapeutic agent is selected from: bone morphogenetic factor, transforming growth factor-beta (TGF-beta), interleukin-1 (IL-1) inhibitors, including but not limited to IL -1ra and its derivatives and Kineret ^™ , anakinra; TNFα inhibitors, including but not limited to soluble TNFα receptor, Enbrel ^™ , etanercept, anti-TNFα antibody, Remicade ^™ , infliximab, and D2E7 antibody; parathyroid hormone and its analogs; parathyroid hormone-related protein and its analogs; E-series prostaglandins; bisphosphonates (such as alendronate and others); bone-enhancing minerals such as fluoride and calcium; non-steroidal anti-inflammatory drugs ( NSAIDs), including but not limited to COX-2 inhibitors such as Celebrex ^TM , Celecoxib and Vioxx ^TM , rofixixime; immunosuppressants such as methotrexate or leflunomide, serine protease inhibitors, Including but not limited to secretory leukocyte protease inhibitor (SLPI); IL-6 inhibitors (including but not limited to anti-IL-6 antibodies), IL-8 inhibitors (including but not limited to anti-IL-8 antibodies), IL- 18 inhibitors (including but not limited to IL-18 binding protein and anti-IL-18 antibodies), interleukin-1 converting enzyme (ICE) modulators; fibroblast growth factors FGF-1 to FGF-10 and FGF modulators; PAF Antagonists; keratinocyte growth factor (KGF), KGF-related molecules, and KGF modulators; matrix metalloproteinase (MMP) modulators; nitric oxide synthase (NOS) modulators, including but not limited to, inducible NOS modulators of glucocorticoid receptors; modulators of glutamate receptors; modulators of lipopolysaccharide (LPS) levels; and norepinephrine and modulators and their mimetics.

In some embodiments, acceptable formulation materials are preferably used in dosages and concentrations that are not toxic to recipients.

In some embodiments, the pharmaceutical composition may contain ingredients for changing, maintaining or retaining, for example, pH, osmotic pressure, viscosity, clarity, color, isotonicity, odor, sterility, stability, dissolution or release rate, Formulation material for absorption or penetration of the composition. In some embodiments, suitable formulation materials include, but are not limited to, amino acids (such as glycine, glutamine, asparagine, arginine, or lysine); antimicrobial agents; antioxidants (such as ascorbic acid, sodium sulfite, or sodium bisulfate); buffers (e.g. borate, bicarbonate, Tris-HCl, citrate, phosphate or other organic acids); bulking agents (e.g. mannitol or glycine); chelating agents (e.g. amine tetraacetic acid (EDTA)); complexing agents (such as caffeine, polyvinylpyrrolidone, β-cyclodextrin, or hydroxypropyl-β-cyclodextrin); fillers; monosaccharides; disaccharides; and other carbohydrates ( such as glucose, mannose, or dextrin); proteins (such as serum albumin, gelatin, or immunoglobulins); colorants, flavoring agents, and diluents; emulsifiers; hydrophilic polymers (such as polyvinylpyrrolidone); Molecular weight polypeptides; salt-forming counterions (e.g., sodium); preservatives (e.g., benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine , sorbic acid or hydrogen peroxide); solvents (such as glycerin, propylene glycol, or polyethylene glycol); sugar alcohols (such as mannitol or sorbitol); suspending agents; surfactants or wetting agents (such as Pluronic class, PEG, sorbitan esters, polysorbates, such as polysorbate20, polysorbate80, triton, tromethamine, lecithin, cholesterol, tyloxapol), stability enhancers (such as sucrose or sorbitol); tonicity enhancers (such as alkali metal halide, preferably sodium or potassium chloride, mannitol, sorbitol); delivery vehicle; diluent; excipient and/or pharmaceutical adjuvant (Remington's Pharmaceutical Science, 18th edition, edited by A.R. Gennaro , Mack Publish Company (1990)).

In some embodiments, the anti-OPGL antibody and/or therapeutic molecule is linked to a half-life extending excipient known in the art. Such excipients include, but are not limited to, Fc domains, polyethylene glycol and dextran. Such excipients are described, for example, in US Application Serial No. 09/428082 and Published PCT Application No. WO99/25044, which are incorporated herein by reference for any purpose.

In some embodiments, one skilled in the art determines the optimal pharmaceutical composition based on, for example, the desired route of administration, mode of delivery, and desired dosage. See, eg, Remington's Pharmaceutical Science, supra. In some embodiments, such compositions can affect the physical state, stability, rate of in vivo release and rate of in vivo clearance of the antibodies of the invention.

In some embodiments, the primary excipient or carrier in the pharmaceutical composition may be aqueous or non-aqueous in nature. For example, in some embodiments, a suitable excipient or carrier may be water for injection, physiological saline, or artificial cerebrospinal fluid, possibly supplemented with other materials commonly used in compositions for parenteral administration. In some embodiments, neutral buffered saline or saline mixed with serum albumin are other exemplary excipients. In some embodiments, the pharmaceutical composition contains Tris buffer at about pH 7.0-8.5, or acetate buffer at about pH 4.0-5.5, which may further contain sorbitol or a suitable substitute thereof. In some embodiments, compositions containing an anti-OPGL antibody, with or without at least one additional therapeutic agent, can be prepared for storage by mixing a selected composition having a desired degree of purity with optional formulation agents ( Remington's Pharmaceutical Science, supra), in the form of a lyophilized cake or an aqueous solution. Furthermore, in some embodiments, compositions comprising an anti-OPGL antibody, with or without at least one additional therapeutic agent, can be formulated as a lyophilizate using a suitable excipient, such as sucrose.

In some embodiments, pharmaceutical compositions of the invention may be selected for parenteral administration. In some embodiments, pharmaceutical compositions may be selected for inhalation or delivery through the alimentary canal, eg, orally. The preparation of such pharmaceutically acceptable compositions is well known in the art.

In some embodiments, formulation ingredients are present in concentrations acceptable to the point of administration. In some embodiments, buffers are used to maintain the composition at physiological pH or slightly lower pH, generally in the pH range of about 5 to about 8.

In some embodiments, when parenteral administration is involved, the therapeutic composition may be pyrogen-free in a pharmaceutically acceptable excipient, parenterally acceptable, containing the desired anti-OPGL antibody, with or without additional therapeutic in the form of an aqueous solution of the agent. In some embodiments, the vehicle for parenteral injection is sterile distilled water in which the anti-OPGL antibody is formulated as a sterile isotonic solution with or without at least one additional therapeutic agent and stored as appropriate. In some embodiments, manufacturing involves formulating desired molecules and agents, such as injectable microspheres, biodegradable particles, polymeric compounds (such as polylactic acid or polyglycolic acid), beads, or liposomes, which can provide the Controlled or sustained release, then delivered by extended-acting injection. In some embodiments, hyaluronic acid may also be used, which has the effect of promoting retention in circulation. In some embodiments, the desired molecule can be introduced using an implantable drug delivery device.

In some embodiments, pharmaceutical compositions may be formulated for inhalation. In some embodiments, an anti-OPGL antibody with or without at least one additional therapeutic agent can be formulated as a dry powder for inhalation. In some embodiments, solutions for inhalation containing anti-OPGL antibodies with or without at least one additional therapeutic agent may also be formulated with a propellant for aerosol delivery. In some embodiments, the solution can be sprayed. Pulmonary administration is further described in PCT Application No. PCT/US94/001875, which describes the pulmonary delivery of chemically modified proteins.

In some embodiments, formulations that can be administered orally are involved. In some embodiments, the anti-OPGL administered in this manner, with or without at least one additional therapeutic agent, may be formulated with or without those carriers conventionally used in the compounding of solid dosage forms such as tablets and capsules. Antibody. In some embodiments, capsules can be designed to release the active portion of the formulation at a point in the digestive tract where bioavailability is maximized and prior systemic degradation is minimized. In some embodiments, at least one additional agent may be included to facilitate absorption of the anti-OPGL antibody and/or any additional therapeutic agent. In some embodiments, diluents, flavoring agents, low melting point waxes, vegetable oils, lubricants, suspending agents, tablet disintegrating agents and binders may also be used.

In some embodiments, the pharmaceutical composition may contain an effective amount of an anti-OPGL antibody, with or without at least one additional therapeutic agent, in admixture of non-toxic excipients suitable for tablet manufacture. In some embodiments, single-dose solutions can be prepared by dissolving the tablet in sterile water, or other suitable excipient. In some embodiments, suitable excipients include, but are not limited to, inert diluents, such as calcium carbonate, sodium carbonate or bicarbonate, lactose, or calcium phosphate; or binders, such as starch, gelatin, or acacia; or lubricants such as magnesium stearate, stearic acid, or talc.

Additional pharmaceutical compositions will be apparent to those skilled in the art, including formulations involving sustained or controlled release delivery formulations containing the anti-OPGL antibody, with or without at least one additional therapeutic agent. In some embodiments, formulation techniques for a variety of other sustained or controlled release delivery modes, such as liposomal carriers, biodegradable microparticles or porous beads, and extended-acting injections, are well known to those skilled in the art . See, eg, PCT Application No. PCT/US93/00829, which describes the controlled release of porous polymeric microparticles for the delivery of pharmaceutical compositions. In some embodiments, sustained release formulations may contain a semipermeable polymer matrix in the form of a shaped product, such as a membrane, or microcapsules. Sustained release matrices can include polyesters, hydrogels, polylactides (US3773919 and EP058481), copolymers of L-glutamic acid and γ-ethyl-L-glutamic acid (Sidman et al., Biopolymers, 22:547 -556 (1983)), poly(2-hydroxyethyl-methacrylate) (Langer et al., J. Biomed. Mater. Res. 15: 167-277 (1981) and Langer, Chem. Tech., 12: 98-105 (1982)), ethylene vinyl acetate (Langer et al., supra) or poly-D(-)-3-hydroxybutyric acid (EP133988). In some embodiments, sustained release compositions may also include liposomes, which can be prepared by several methods well known in the art. See, eg, Eppstein et al., Proc. Natl. Acad. Sci. USA, 82:3688-3692 (1985); EP036676; EP088046 and EP143949.

Generally, pharmaceutical compositions for in vivo administration are sterile. In some embodiments, this can be achieved by filtration through sterile filtration membranes. In some embodiments, where the composition is lyophilized, this method can be used to sterilize either before or after lyophilization and reconstitution. In some embodiments, compositions for parenteral administration may be stored in lyophilized form or in solution. In some embodiments, parenteral compositions are typically placed in containers with sterile outlets, such as intravenous solution bags or vials with caps piercable by a hypodermic needle.

In some embodiments, once the pharmaceutical composition has been formulated, it can be stored in sterile vials as a solution, suspension, gel, emulsion, solid, or as a dehydrated or lyophilized powder. middle. In some embodiments, such formulations may be stored in a ready-to-use form or in a form reconstituted (eg, lyophilized) prior to administration.

In some embodiments, the invention relates to kits for the preparation of single dosage administration units. In some embodiments, each kit can include a first container containing dry protein and a second container containing an aqueous formulation. In some embodiments of the invention, kits comprising single or multi-lumen prefilled syringes (eg, liquid syringes and dissolution syringes) are included.

In some embodiments, the effective amount of a pharmaceutical composition containing an anti-OPGL antibody with or without at least one additional therapeutic agent to be used therapeutically depends on, for example, the context of treatment and the subject. Those skilled in the art will appreciate that, according to some embodiments, the appropriate dosage level for treatment will therefore depend in part on the molecule being delivered, the indication for which the anti-OPGL antibody is being used with or without at least one additional therapeutic agent, the route of administration, and the size of the patient. (weight, body surface area or organ size) and/or condition (age and general health). In some embodiments, the physician can adjust the dosage and alter the route of administration to obtain the optimum therapeutic effect. In some embodiments, typical dosages may range from about 0.1 microgram/kg up to about 100 mg/kg or more, depending on the factors mentioned above. In some embodiments, the dose may be 0.1 microgram/kg up to about 100 mg/kg; or 1 microgram/kg up to about 100 mg/kg; or 5 microgram/kg up to about 100 mg/kg.

In some embodiments, the frequency of dosing takes into account the pharmacokinetic parameters of the anti-OPGL antibody and/or any additional therapeutic agent in the formulation used. In some embodiments, the physician administers the composition until a dose is reached to achieve the desired effect. In some embodiments, it may thus be administered in a single dose, or as two or more doses over time (which may or may not contain the same amount of the desired molecule), or as a continuous infusion via an implanted device or catheter. to apply the composition. The precise appropriate dosage is routinely determined by those skilled in the art, and this is a routine task for them ordinarily. In some embodiments, the appropriate dosage can be ascertained through the use of appropriate dose-response data.

In some embodiments, the route of administration of the pharmaceutical composition is a known method, for example, oral, intravenous injection, intraperitoneal, intracerebral (intraparenchymal), intraventricular, intramuscular, intraocular, intraarterial, porta hepatic Intra-, or intralesional routes; by sustained-release systems or by implanted devices. In some embodiments, the composition may be administered by bolus injection or continuous infusion, or by an implanted device.

In some embodiments, the composition may be administered topically by implanting a membrane, sponge or other suitable material onto which the desired molecule is adsorbed or encapsulated. In some embodiments, where implantable devices are administered, the device can be implanted in any suitable tissue or organ, and delivery of the desired molecule can be by diffusion, time-released bolus, or continuous administration.

In some embodiments, it is desirable to use a pharmaceutical composition comprising an anti-OPGL antibody, with or without at least one additional therapeutic agent, ex vivo. In such cases, cells, tissues and/or organs from the patient are contacted with a pharmaceutical composition containing an anti-OPGL antibody, with or without at least one additional therapeutic agent, and then these cells, tissues and/or organs are subsequently implanted back to the patient.

In some embodiments, the anti-OPGL antibody and/or any additional therapeutic agent can be delivered by implanting cells genetically engineered to express and secrete the polypeptide using the methods described herein. In some embodiments, such cells may be animal cells or human cells, and may be autologous, allogeneic, or xenogeneic. In some embodiments, cells can be propagated indefinitely. In some embodiments, cells can be encapsulated to avoid infiltration of surrounding tissue in order to reduce the chance of an immune response. In some embodiments, the capsule material is generally a biocompatible semipermeable polymer shell or membrane that allows the release of the protein product but prevents the patient's immune system or other deleterious agents from surrounding tissues from destroying the cells.

Example

The following examples, including experiments performed and results obtained, are provided for the purpose of illustration only and are not intended to limit the invention.

Example 1

Cloning of αOPGL-1 heavy and light chains

Transgenic mice containing human immunoglobulin genes were immunized with CHO cells expressing full-length human OPGL cDNA. Lymph nodes from immunized mice were fused with murine myeloma cells to generate hybridomas. Hybridoma cell line supernatants were assayed for antibodies reactive with human OPGL in an ELISA assay. It was found that the anti-OPGL expressing hybridoma cell lines AMG6.5, AMG6.4, and AMG6.1 expressed antibodies with high affinity to OPGL (Kd were 0.28nM, 0.29nM, and 0.23nM), and AMG6.5 was selected to clone. The heavy and light chain cDNA clones from AMG6.5 and AMG6.4 were identical, and AMG6.5 was used to clone the αOPGL-1 light chain cDNA while AMG6.4 was used to clone the αOPGL-1 heavy chain cDNA.

Cloning of αOPGL-1 light chain

The αOPGL-1κ light chain variable region was obtained from the first cDNA strand prepared from AMG6.5 total RNA by PCR amplification method. A random primer with an extended 5'-linker (5'-GGCCGGATAGGCCTCACNNNNNNNT-3' (SEQ ID NO: 15)) was used, and the Gibco SuperScript II ^™ Preamplification System for FirstStrand cDNA Synthesis Kit (cat.no.18089-011 ) to prepare the first strand cDNA from AMG6.5 total RNA using the materials and methods provided. The following oligonucleotides were used for PCR:

5′κ RACE Primer:

5'-GAT GAC CCA GTC TCC AGC CAC CCT G-3' (SEQ ID NO: 5)

3'κRACE Primer:

5'-AAG GGT CAG AGG CCA AAG GAT GG-3' (SEQ ID NO: 6)

Amplified DNAs were cloned into pCRII-TOPO (Invitrogen) and the resulting plasmids were sequenced. Primers for full-length αOPGL-1 κ chain PCR amplification were designed using the κ chain consensus sequence. The 5'αOPGL-1κ primer was inserted into the Xbal site for cloning (TCTAGA) and the "CCACC" Kozak sequence before the start Met codon. The 3'αOPGL-1κ primer was inserted into the Sa/I site after the stop codon for cloning.

5′αOPGL-1κ primer:

5'-CAA C TC TAG A CC ACC ATG GAA ACC CCA GCG-3' (SEQ ID NO: 7)

XbaI site Kozak M E T P A (SEQ ID NO: 16)

3′αOPGL-1κ primer:

5'-TTT GAC GTC GAC TTA TCA ACA CTC TCC CCT GTT GAAG-3' (SEQ ID NO: 8)

SalI site * * C E G R NF (SEQ ID NO: 17)

A full-length αOPGL-1κ chain cDNA clone was obtained by PCR amplification using 5' or 3' αOPGL-1κ primers using the AMG6.5 first cDNA strand as described above. The PCR reaction generated a 738 bp fragment encoding 235 amino acid residues of the αOPGL-1 κ chain (including the 20 amino acid κ chain signal sequence) ( FIG. 4 , SEQ ID NO: 4). This fragment was used to construct a kappa light chain expression vector after purification using the QIAquick PCR purification kit (Qiagen cat. no. 28104).

The 738bp full-length κ fragment generated above was digested with Xbal and SalI, purified using the PromegaWizard DNA Clean-Up system (Promega cat.no.A7100), and cloned into pDSRα19 to generate plasmid αOPGL-1-κ/pDSRα19 (Figure 5) . pDSR[alpha]19 has been described previously (see WO90/14363, which is hereby incorporated by reference for any purpose (see, eg, Figure 12)). Briefly, to make pDSRα19, pDSRα2 was modified as follows: shortened FSH polyA from approximately 1400 bp to 885 bp, now ending at the Ndel site; dihydrofolate reductase (DHFR) promoter It now contains 209 base pairs, shortened by about 1 kb from the 5' end; the Bglll fragment is missing about 550 base pairs from the DHFR polyA sequence.

Sequencing of the alpha OPGL-1 kappa light chain expressing clone confirmed that it encodes the same peptide identified in the AMG6.5 hybridoma. The final expression vector αOPGL-1-κ/pDSRα19 is 5476bp and contains 7 functional regions shown in Table 2.

Table 2: Characteristics of αOPGL-1-κ/pDSRα19

Plasmid base pair number

2-881

Transcription termination/polyadenylation signal from the alpha-subunit of the bovine pituitary glycoprotein hormone (α-FSH) (Goodwin et al., Nucleic Acids Res. 198311:6873-82; Genbank Accession No. X00004)

882-2027

Mouse dihydrofolate reductase (DHFR) minigene containing endogenous mouse DHFR promoter, cDNA coding sequence, and DHFR transcription termination/polyadenylation signal (Gasser et al., Proc Natl Acad Sci USA198279:6522-6; Nunberg et al., Cell 198019:355-64; Setzer et al., J Biol Chem.1982257:5143-7; McGrogan et al., J Biol Chem.1985260:2307-14)

2031-3947

pBR322 sequence containing the ampicillin resistance marker gene and origin of plasmid replication in Escherichia coli (Genbank Accession No. J01749)

3949-4292

SV40 early promoter, enhancer and origin of replication (Takebe et al., Mol Cell Biol 19888:466-72, Genbank Accession No. J02400)

4299-4565

Translational enhancer element from HTLV-1 LTR domain (Seiki et al., Proc Natl Acad Sci USA198380:3618-22, Genbank Accession No. J02029)

4574-4730

Intron from SV4016S, 19S splice donor/acceptor signal (Okayama and Berg, Mol Cell Biol 19833:280-9, Genbank Accession No. J02400)

4750-5476

αOPGL-1κ light chain cDNA between Xbal and Sa/I sites.

Circular plasmid map of the vector shown in Figure 5.

Cloning of αOPGL-1 heavy chain

[alpha]OPGL-1IgG2 heavy chain was cloned from AMG6.4 hybridoma double-stranded cDNA prepared with Clontech Marathon ^(TM) cDNA Amplification Kit (cat no. K1802-1). Rapid 5' and 3' amplification of cDNA ends by using human germline IgG2 heavy chain constant region-specific primers (shown below) and RACE primers and other materials and methods provided in the Marathon ^™ cDNA Amplification Kit (RACE) technology to achieve the amplification of AMG6.4 heavy chain cDNA.

5'IgG2RACE Primer

5'-GGC ACG GTC ACC ACG CTG CTG AG-3' (SEQ ID NO: 9)

3'IgG2RACE Primer

5'-CCT CCA CCA AGG GCC CAT CGG TCT-3' (SEQ ID NO: 10)

The 600bp 5'RACE product and the 1200bp 3'RACE product were cloned into pCR2.1 (Invitrogen) and sequenced. This sequence information was used to design αOPGL-1 heavy chain-specific primers for cloning the full-length sequence. The heavy chain 5' primer (5'αOPGL-1Ig G2 primer) was directed against the sense strand and had a HindIII site and a consensus Kozak sequence before the native start site. The heavy chain 3' primer (3'αOPGL-1 IgG2 primer) is an antisense primer and contains a SalI site and a stop codon after the last amino acid of the heavy chain IgG2 sequence.

5′αOPGL-1 IgG2 Primer:

5′-CAG AAGCTT GA CCACC ATG GAG TTT GGG CTG AGC TGG CTT TTT CTT GTG GC-3′

(SEQ ID NO: 11)

HindIII Kozak M E F G L S W L F L V A

(SEQ ID NO: 18)

3′αOPGL-1 IgG2 Primer:

5'-GCA T GTCGAC TTA TCA TTT ACC CGG AGA CAG GGA GAG-3' (SEQ ID NO: 12)

SalI＊＊K G P S L S L (SEQ ID NO: 19)

The double-stranded cDNA above was used to generate full-length heavy chain cDNA by PCR amplification using 5'- and 3'-αOPGL-1 IgG2 primers. PCR generated a 1433 bp fragment encoding 467 amino acid residues of the αOPGL-1 IgG2 heavy chain protein (including the 19 amino acid IgG signal sequence) ( FIG. 2 , SEQ ID NO: 2). After purification using the QIAquick PCR purification kit (Qiagencat no. 28104), this fragment was used to construct a heavy chain expression vector as follows.

The DNA encoding the full-length IgG2 heavy chain fragment generated above was digested with HindIII and SalI, purified using the QIAquick gel extraction kit (Qiagen cat no. 28704), and the fragment was cloned into pDSRα19. The resulting expression plasmid was called αOPGL-1-IgG2/pDSRα19 (Fig. 6). All vector components were identical to the αOPGL-1-κ/pDSRα19 vector above, except that the αOPGL-1-IgG2 heavy chain cDNA replaced the αOPGL-1κ light chain cDNA between the Xbal and SalI sites. Sequencing of the αOPGL-1-IgG2 heavy chain expressing clone confirmed that it encodes the same polypeptide identified in the AMG6.4 hybridoma.

Example 2

αOPGL-1 expression in CHO cells

By co-transfecting αOPGL-1-κ/pDSRα19 and αOPGL-1-IgG2/pDSRα19 into dihydrofolate reductase-deficient (DHFR ⁻ ) Chinese hamster ovary cells (CHO AM-1/D, US Patent No. 6210924) followed by Individual clones were isolated and analyzed to achieve stable expression of the αOPGL-1 antibody.

On the day before transfection (day 0), grow in CHO d ^- medium (DMEM-high glucose, 10% fetal bovine serum, 1% penicillin/streptomycin/glutamine, 1X sodium pyruvate, 1% Non-Essential Amino Acids (NEAA) and 1% h t supplement 1.5x10 ⁶ AM-1/D cells in ) were plated in 100 mm tissue culture dishes. On the first day, 400 μl of serum-free RPMI1640 medium Aliquot into 12x75mm polypropylene test tubes. Add 24 microliters dropwise to the medium - LT1 reagent (Mirus Corporation) and the mixture was incubated at room temperature for 10 minutes. A total of 15 micrograms of linearized plasmid DNA (7.5 micrograms of αOPGL-1-κ/pDSRα19 and 7.5 micrograms of αOPGL-1-IgG2/pDSRα19 digested with Pvu1) were then added dropwise to the mixture and incubated at room temperature for 10 minutes.

Remove CHO d-medium from cells with 10 ml Dulbecco's phosphate-buffered saline washing. Add 6 mL to cells supplemented with HT, L-glu, NEAA and sodium pyruvate serum-free MEM medium. Add the DNA/LT1 complex dropwise to the plate and shake carefully back and forth to evenly distribute the DNA to the cells. After 6 hours of incubation in the tissue culture incubator, the medium was replaced with fresh CHO d-medium. After 48 hours, cells were split into 10 CHO selection media (DMEM-high glucose, 10% dialyzed fetal bovine serum, 1% penicillin/streptomycin/glutamine, 1% non-essential amino acids and 1X pyruvate sodium) 100mm Petri dish. The medium was changed twice a week until colonies appeared.

After 10-14 days, use 1x trypsin-EDTA 5mm cloning dish Colonies were picked and grown in 24-well tissue culture plates with CHO selection medium. When the cells became confluent, serum-free medium (CHO selection medium, FBS negative) was added and harvested after 48 hours. These conditioned media were analyzed for antibody expression by Western blotting with a horseradish peroxidase (HRP)-conjugated goat anti-human IgG Fc antibody (Pierce, Rockford, IL) to determine the αOPGL-1 heavy chain, and αOPGL-1 light chain was assayed with goat anti-human kappa chain antibody (Pierce, Rockford, IL) followed by HRP-conjugated rabbit anti-goat IgG (H+L antibody) (Pierce, Rockford, IL). The highest expressing clone was expanded and stored in liquid nitrogen.

Example 3

Preparation of αOPGL-1

Preparation and generation of cell line 125Q

CHO cells producing αOPGL-1 were cloned by two rounds of limiting dilution in 96-well plates under serum-free conditions. Clones were selected based on production and growth characteristics in various suspension vessels. EIA was performed to select for clones producing the highest levels of [alpha]OPGL-1. Growth characteristics, including doubling time and density, were then determined by growing the clones in 100 ml, 250 ml, 500 ml, 1 liter and 3 liter spinner flasks and in 3 liter Aplkon bioreactors. The clone that reached the highest density with the fastest doubling time in culture was selected and labeled as cell line 125Q. When clonal expansion has yielded enough cells to freeze 360 ampoules at approximately ^1x107 cells/ml, resuspend the cells in refrigerated serum-free medium supplemented with 10 ml/L non-essential amino acids and 10 ml/L L-glutamine (Gibco/LTI/Invitrogen), and 10% dimethylsulfoxide (JT Baker) in 90% VM-Soy Batch medium (see Table 3 for details)) and frozen. Store ampoules in restricted access equipment and submerge in liquid nitrogen in liquid nitrogen vacuum flasks.

The 125Q cell line was selected based on the cell line used to produce αOPGL-1 based on growth and production in small-scale rotators and larger-scale bioreactors.

cell culture

αOPGL-1 was prepared by expression in the 125Q cell line, a clonal line of CHO cells expressing αOPGL-1 from the plasmids αOPGL-1-κ/pDSRα19 and αOPGL-1-IgG2/pDSRα19. Figure 19 shows the cell culture method for αOPGL-1. For each round of preparation, cells from vials of the 125Q cell line were initially supplemented in 50 mL with 10 mL/L non-essential amino acids and 10 mL/L L-glutamine (Gibco/LTI/Invitrogen) (VM-Soy Supp) The VM-Soy Batch medium (see Table 3 for the composition) was cultured in a 125 ml Erlenmeyer flask at 100 rpm for 5 days. The whole culture was then used to inoculate VM-Soy Supp in a 500 ml spinner flask to 3×10 ⁵ viable cells/ml (3E5vc/ml), and spin culture at 70 rpm for 3-4 days. The whole culture from the 500 ml spinner flask was used to inoculate VM-SoySupp in a 3L spinner flask to 3E5vc/ml and spin culture at 70 rpm for 3-4 days.

The culture in the 3L spinner flask was then pipetted into two 3L spinner flasks to 3E5vc/ml in VM-Soy Supp without phenol red and grown under the same conditions. These spinner flask cultures were then used to inoculate four other spinner flasks to 3E5vc/ml in VM-Soy Supp without phenol red and grown under the same conditions. Four liters of culture from four 3L spinner flasks were used to inoculate 10 L of VM-Soy Supp without phenol red in a 20 L bioreactor run in batch feed mode for 7-10 days. In batch feeding mode, a nutrient feed containing concentrated medium ("Feed" is indicated in Table 3 below) was added to maintain cell growth and culture survival.

The whole culture from the 20L bioreactor was then used to inoculate 70L of VM-Soy Supp without phenol red in a 150L bioreactor and the bioreactor was run in batch feed mode for 9-10 days. Finally, approximately 880 LVM-Soy (without supplement or phenol red) in a 2000 L bioreactor was inoculated with the entire culture from the 150 L bioreactor, and the bioreactor was run in batch feed mode. The feed rate was measured during the batch feed mode such that the glucose level in each bioreactor culture was maintained at 0.6 g/L. Cell density and glucose concentration were determined daily and feed rates adjusted accordingly.

Production in the 2000L bioreactor lasted approximately two weeks, during which time the cells constitutively produced αOPGL-1 and secreted it into the cell culture medium.

The production reactor was controlled by setting pH, temperature and dissolved oxygen content: pH was 7.0 and controlled by carbon dioxide gas and sodium carbonate addition; dissolved oxygen was 120 mmHg and controlled by air, nitrogen and oxygen flow. Cells were maintained at 37°C throughout the process. All gases pass through membrane filters of 0.22 micron or smaller pore size.

At the end of the preparation, the cell culture broth was fed to a disc stack centrifuge and the culture supernatant was separated from the cells. The concentrate was further clarified by passing through a Cuno 90SP depth filter followed by a 0.2 micron Posidyne filter (Pall Co.). The clarified conditioned medium was then concentrated by tangential flow ultrafiltration (UF) using a 50kD NMWL membrane (Millipore Biomax50). Concentrate the conditioned media 15 to 30-fold. The resulting concentrated conditioned medium (CCM) was then processed by purification or frozen for subsequent purification. Figure 19 summarizes the production method.

cell culture medium

The cell culture medium used throughout the cell culture was based on Dulbecco's Modified Eagle's Medium/Ham's Nutrient F12 (DMEM/F12, 1:1) and contained supplementary levels of amino acids, additional nutrients and salts, soy hydrolyzate and recombinant human insulin( Eli Lilly). The composition is listed in Table 3, and the medium was called VM-Soy. The media solution was filtered through a 0.2 micron pore size membrane filter prior to use.

Table 3 Cell culture medium composition

Components of minimal media and feed

Purification method

αOPGL-1 expressed in CHO cells is secreted into the extracellular medium. A series of steps can be applied to prepare pure substances. The method uses hydrophobic charge induction, cation exchange, and hydrophobic interaction chromatography, as well as a low pH step and a virus filter. These methods are described below.

A. Hydrophobic Charge Induction Chromatography (HCIC)

This chromatography step removes most host cell proteins and DNA. Concentrated conditioned medium (CCM) was filtered through a Cuno30SP filter followed by a Cuno VR07 charged cellulose-based filter before loading onto MEP HyperCel resin. After loading, the column was washed with equilibration buffer (20mM Tris pH 7.2). Antibody was eluted from the resin using a low pH buffer (20 mM sodium acetate, pH 5.0). Upon elution from the column, the product was collected based on the absorbance at 280 nm of the column effluent.

B. Virus inactivation

The MEP pool was titrated to pH 3.7 and held for approximately 60 minutes to inactivate possible contaminating retroviruses. After the hold step, the pH was adjusted to approximately 6.0.

C. Virus filtering

The pH adjusted pool was filtered through a Millipore Viresolve NFR filter or equivalent. Antibodies flow through the filter, while possible contaminating viruses ≥ 50 nm remain.

D. Cation Exchange Chromatography (CEX)

Antibodies were further purified by cation exchange chromatography using SP Sepharose HP (Amersham Pharmacia) or equivalent. A cation exchange chromatography step removes additional CHO cell proteins, DNA, low molecular weight proteins, and aggregated forms of αOPGL-1. The virus filtered pool was loaded onto the cation exchange resin, and after loading, the column was washed with equilibration buffer (20mM NaMES pH6.2). The antibody was then eluted with a linear gradient of increasing salt (20 mM NaMES pH 6.2, 0 M NaCl to 20 mM NaMES pH 6.2; 0.3 M NaCl). Upon elution from the column, the product was collected based on the absorbance at 280 nm of the column effluent.

E. Hydrophobic Interaction Chromatography (HIC)

Antibodies were further purified by hydrophobic interaction chromatography using Phenyl Toyopearl 650S (Tosoh Biosep) or equivalent. Hydrophobic interaction chromatography was used as a polishing step and removed additional CHO cell proteins, DNA, low molecular weight proteins, and aggregated forms of αOPGL-1. The cation exchange pool was adjusted to a conductivity >105 mS/cm at 15-25°C by adding ammonium sulfate before loading onto the column. After loading the column was washed with equilibration buffer (1M potassium phosphate, pH 8). Antibody was then eluted with a linear gradient of decreasing salt concentration (1 M potassium phosphate, 0 mM Tris pH 8 to 0 M potassium phosphate, 20 mM Tris pH 8). Upon elution from the column, the product was collected based on the absorbance at 280 nm of the column effluent.

F. Concentration and Diafiltration

The HIC column pool was concentrated by tangential flow ultrafiltration using a 50kD NMWL membrane (Millipore Biomax 50) and diafiltered into formulation buffer. The formulation buffer contained 10 mM acetate, 5% sorbitol, pH 5.2 and 30 mg/mL of αOPGL-1.

Final filtration and storage

The purified product was passed through a 0.2 micron P\/DF filter (Millipore), sampled, and stored in a safety freezer at approximately -30°C.

Example 4

Binding specificity of αOPGL-1

Antibodies produced in CHO cells transfected with two expression vectors as discussed in Examples 1 and 2 can be used in Examples 4, 5, and 6 below.

Human OPG binds and neutralizes OPGL in rats, mice and macaques as well as humans. [alpha]OPGL-1 bound human OPGL with high affinity, but not significantly to murine OPGL (Table 4).

Table 4: Affinity of αOPGL-1 for membrane-expressed human, macaque or mouse sequences of OPGL

OPGL from these species is expressed as a full-length membrane-bound protein in CHO cells. Binding of αOPGL-1 to cell surface expressed OPGL was assessed by FACS analysis of cells incubated with αOPGL-1 and FITC-labeled anti-human IgG2 secondary antibody. αOPGL-1 binds human and macaque OPGL, but does not specifically bind mouse OPGL.

In addition, human OPG has been reported to exhibit weak binding to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) (Truneh et al., 2000), which is a related member of the TNF family, which exhibits DNA binding to OPGL. and amino acid sequence homology (Lacey et al., 1998). However, there was no detectable binding of OPG to other TNF-related proteins such as TNFα, TNFβ, or CD40 ligand.

αOPGL-1 specifically bound OPGL on EIA plates (Fig. 7). Recombinant soluble OPGL (2 μg/ml) was coated onto 96-well EIA plates for 16-24 hours at room temperature. After blocking with 1% BSA in PBS, various concentrations of αOPGL-1 (approximately 2 ng/ml to 1000 ng/ml) diluted in 1% BSA/PBS were added to the wells and the plates were incubated for approximately 2 hours at room temperature. Bound antibodies were detected using goat anti-human IgG (Fab')-HRP using a TMB-H2O2 (tetramethylbenzidine and hydrogen peroxide) substrate mixture. Read absorbance at 450nm and 650nm.

[alpha]OPGL-1 specifically binds to OPGL expressed on the surface of transfected cells (Figure 8). αOPGL-1 (100ng/ml) diluted in FACS buffer (PBS, 0.1% BSA, 0.01% sodium azide) was treated with various concentrations of OPGL, TNFα, TNFβ, TRAIL, or CD40 ligand (approximately 0.1ng /ml to 1000ng/ml) were pre-incubated and then added to approximately 200,000 CHO REN218-9 cells, which are CHO cells stably expressing membrane-bound OPGL on the cell surface. After 1 hour at 2-8°C, unbound antibody was removed by centrifugation and washed. Cells were then incubated with FITC-labeled F(ab')2 goat anti-human IgG (Fcγ fragment specific) for 30 minutes at 2-8°C. After centrifugation and washing, cell surface fluorescence was measured using flow cytometry. Figure 8 demonstrates that the binding of αOPGL-1 to CHO REN218-9 cells is specific and competitively reduced by the addition of soluble OPGL, but not TNFα, TNFβ, TRAIL, or CD40 ligands.

In competition experiments, addition of exogenous OPGL inhibited αOPGL-1 binding to OPGL on EIA plates (FIG. 9), but not addition of TNFα, TNFβ, TRAIL, or CD40 ligand (FIG. 10). This procedure was performed in essentially the same manner as above for the binding of αOPGL-1 to OPGL on EIA plates, but a constant concentration of αOPGL-1 (100 ng/mL) was mixed with various concentrations of Dissolved OPGL or other ligands (approximately 1 ng/ml to 1000 ng/ml each) were pre-incubated.

Example 5

αOPGL-1 neutralizing activity

Inhibition of osteoclast formation

RAW264.7 (ATCC No. TIB-71, Manassas, VA) is a murine macrophage cell line derived from Abelson murine leukemia virus-induced tumors. RAW264.7 cells differentiated into osteoclast-like cells in the presence of OPGL. For a detailed description of the basic analysis of osteoclast production in RAW cell culture formats in the presence of OPGL, see Simonet et al. (1997) Cell 89 p. 309, and Lacey et al. (1998) Cell 93 p. 165, which are hereby incorporated by reference for any purpose.

Ligands stimulate RAW cells to differentiate into osteoclast-like cells, which can be measured by TRAP activity and osteoclast properties. Thus, the effect of αOPGL-1 on osteoclastogenesis can be determined.

In cell culture medium (DMEM, 10% FBS, 0.292 mg/ml L-Glut, 100 units/ml penicillin G, 100 μg/ml streptomycin sulfate), in constant amount of OPGL (40ng/ml) and different amount RAW cells were incubated for 4 days in the presence of αOPGL-1 (6.3ng/ml to 200ng/ml). At the end of the fourth day, cells were stained for tartrate-resistant acid phosphatase (TRAP) activity assay by osmosis and acidification followed by 5 min treatment with p-nitrophenyl phosphate. Briefly, cells were aspirated from the medium, 100 microliters of citrate buffer (410 mL 0.1M citric acid, 590 mL 0.1M citrate, trisodium salt, 1 mL tritonX-100) was added to each well, and Plates were incubated for 3-5 minutes at room temperature. Then add 100 microliters of PNPP solution (157.8 mg acid phosphatase reagent (Sigma 104100), 7.2 ml tartrate solution (Sigma cat.no.387-3), and 22.8 ml citrate buffer), and place the plate at room temperature Incubate for 3-5 minutes. The reaction was terminated by adding 50 μl of 0.5 M NaOH solution.

TRAP converts p-nitrophenyl phosphate to p-nitrophenol, quantified by densitometry at 405 nm. Thus TRAP activity, which is a surrogate marker for osteoclast development, correlates with optical density at 405 nm. Figure 11 shows a graph of optical density versus [alpha]OPGL-1 concentration and demonstrates that [alpha]OPGL-1 inhibits osteoclastogenesis in this assay.

Inhibition of OPGL binding to its receptor

The efficacy of αOPGL-1 is demonstrated by its ability to block the binding of OPG ligands to its cognate receptor, the osteoclast differentiation and activation receptor (ODAR, also known as RANK). This assay employs homogeneous time-resolved fluorescence resonance (HTRF) to measure the binding of αOPGL-1 to europium-conjugated osteoprotegerin ligand (Eu-OPGL). If αOPGL-1 inhibits Eu-OPGL binding to ODAR, the fluorescence output will decrease, and the amount of αOPGL-1 present is inversely proportional to the amount of fluorescence.

OPGL was labeled with europium, which emits at 620 nm when excited with 337 nm light. ODAR is fused to FLAG and fused to Fc, the Fc-ODAR-FLAG fusion protein is labeled with an anti-FLAG antibody linked to allophycocyanin (APC), a fluorophore that emits at 665nm when excited at 620nm light . Thus, when Eu-labeled OPG ligand binds the Fc-ODAR-FLAG/anti-FLAG-APC complex, the ternary complex emits at 665 nm when excited with 337 nm light.

0.05 μg/ml Eu-OPGL with various concentrations (0.1 to 150 ng/ml) of αOPGL-1 in assay buffer (50 mM Tris pH8, 100 mM NaCl, 0.05% NaN ₃ , 1% BSA, and 0.05% Tween20) at room temperature Incubate for approximately 1 hour at 100°C (pre-incubation mixture). A mixture of Fc-ODAR-FLAG (1 μg/ml) and anti-FLAG-APC (2.5 μg/ml) was also prepared in assay buffer and incubated for 1 hour at room temperature (fluorochrome mixture). Equal volumes of the preincubation mixture and the fluorochrome mixture were then combined and incubated at room temperature for 3 hours. Fluorescence was determined by plate reading on a Packard Discovery HTRF microplate analyzer using an excitation wavelength of 337 nm and an emission wavelength of 665 nm.

When αOPGL-1 was pre-incubated with Eu-OPG ligand and then mixed with Fc-ODAR-FLAG/anti-FLAG-APC, the fluorescence intensity at 665nm decreased in a dose-dependent manner, as shown in Figure 12, proving that αOPGL162 can effectively Inhibits the binding of OPGL to ODAR.

Example 6

Pharmacokinetics in macaques

Six male and six female macaques, aged less than 4.5 years and weighing 2-4 kg, were divided into 4 dose groups. Group 1 consisted of three males and three females. Groups 2, 3, and 4 each consisted of one male and one female. Animals in Group 1 were administered a single SC dose of 1 mg/kg αOPGL-1, while animals in Groups 2, 3, and 4 were administered a single IV dose of 0.1, 1.0, or 10.0 mg/kg αOPGL-1, respectively.

Animals received [alpha]OPGL-1 expressed by transfected Chinese Hamster Ovary (CHO) cells. Serum samples were taken for the determination of αOPGL-1 levels, antibody analysis, bone renewal marker serum N-telopeptide (serum N-Tx) analysis, alkaline phosphatase (ALP) analysis, and serum calcium (serum Ca) analysis. Urine was collected for analysis of N-telopeptide (urinary N-Tx) and creatinine.

Serum concentration-time profiles after IV administration were characterized by a three-phase distribution (Figure 13). Initially, there was a rapid distribution period followed by a significantly slower plateau, which appeared to be concentration-dependent. The third observation period is the rapid elimination period.

Apply non-compartmental analysis of complete serum concentration-time curves using WinNonlin Professional (v1.5), and index analysis of data up to 14 days after test substance administration, and 10,000 ng/mL using SAAM II (v1.1.2) The above analysis was used to study the pharmacokinetics of αOPGL-1 in monkeys. The average initial volume of distribution for all IV doses was 28.9 ml/kg, similar to plasma concentrations. The volume at steady state (Vss) for all IV dosing distributions averaged 39 ml/kg. Index analysis showed that αOPGL-1 had an average distribution half-life (t _1/2α ) of 6.02 hours. In the second phase of continuation, the half-life (t _1/2β ) was extended from 86.9 hours at 0.1mg/kg dose to 444 hours at the 10.0 mg/kg dose. The non-compartmental estimated terminal elimination half-life (t _1/2z ) averaged 31 hours for all IV dosing groups. It was found that the clearance rate (CL, CL/F) of αOPGL-1 was non-linear, and the average clearance rate of animals receiving 10mg/kg dose of IV administration was 0.120ml/hr/kg) than that receiving 0.1mg/kg (0.401ml/hr /kg) is 3.3-fold lower.

Following subcutaneous administration, absorption is slow, with an average peak concentration ( _Cmax ) of 11,600 ng/ml at 132 hours. The range of exposures following SC administration was highly variable, resulting in a mean clearance of 0.387±0.281 ml/hr/kg and a mean residence time of 202±80.1 hours. The average bioavailability is 89%.

The preceding data are summarized in Table 5.

Table 5: Mean (± SD) non-compartmental pharmacokinetic parameters in rhesus monkeys following IV and SC administration of a single dose of αOPGL-1 ^a

^a for the three values reported with significance plots

^b Not determined, PK samples end in plateau (beta) phase, so terminal phase was not observed

^c not applicable

αOPGL-1 caused a rapid decrease in serum N-Tx levels within 24 hours after administration ( FIG. 14 ). The mean time to onset of maximum effect was found to be between 12 hours and 7 days after IV dosing was increased from 0.1 to 10 mg/kg, and between 12 hours and 11 days for animals receiving SC doses of 1.0 mg/kg. The maximal effect increased approximately 80 to 91% with doses ranging from 0.1 to 1 mg/kg. However, a maximum inhibition of 91% was observed at the higher dose without further inhibition. Mean serum N-Tx levels returned to baseline 28 days after IV administration of 0.1 mg/kg and 70 days after SC administration of 1 mg/kg. Urine N-Tx showed a similar trend to serum N-Tx, with all groups returning to baseline values except for the study day 105 (Figure 15).

Seven days after IV dosing of 10.0 mg/kg serum Ca suppression increased with dosing to a mean nadir of 31.6% below the baseline mean. All other dosing groups had a mean decrease in serum Ca of less than 26.4% of their baseline mean. On day 17, serum Ca levels of all treated animals returned to within 10% of their baseline mean (Fig. 20).

Because bone resorption and formation are closely related, changes in the bone formation marker (ALP) were also observed to decline at ALP levels much more slowly and for a longer period of time compared to the formation marker N-Tx (Fig. 21 ). The observed decrease in markers of bone formation (ALP) prior to bone resorption markers following αOPGL-1 administration demonstrates that αOPGL-1 is a bone anti-resorptive agent.

Most animals (9 out of 12) developed anti-αOPGL-1 antibodies. The incidence of anti-αOPGL-1 antibodies was not dose- or route-dependent. It was not possible to assess the effect of anti-αOPGL-1 antibody on the pharmacokinetics of αOPGL-1 above 0.1 mg/kg when there were no dosing groups with antibody-negative and positive animals. At 0.1 mg/kg IV, most of αOPGL-1 was cleared before antibody production, so no effect on αOPGL-1 treatment was observed ( FIG. 16 ).

Claims (51)

1. an antibody, comprises heavy chain and light chain, wherein:

A) heavy chain comprises the variable region shown in the aminoacid sequence of SEQ ID NO:13; And

B) light chain comprises the variable region shown in the aminoacid sequence of SEQ ID NO:14; And

Wherein said antibody and osteoprotegerin ligand (OPGL) interact.

2. the antibody of claim 1, it comprises heavy chain, described heavy chain comprise SEQ ID NO:2 from residue 20 to the aminoacid sequence of residue 467.

3. the antibody of claim 1, it comprises heavy chain, described heavy chain forming from residue 20 to the aminoacid sequence of residue 467 by SEQ ID NO:2.

4. the antibody any one of claim 1-3, it comprises light chain, described light chain comprise SEQ ID NO:4 from residue 21 to the aminoacid sequence of residue 235.

5. the antibody any one of claim 1-3, it comprises light chain, described light chain forming from residue 21 to the aminoacid sequence of residue 235 by SEQ ID NO:4.

6. the antibody of claim 1, it comprises heavy chain and light chain, wherein said heavy chain comprise SEQ ID NO:2 from residue 20 to the aminoacid sequence of residue 467, and wherein said light chain comprise SEQ ID NO:4 from residue 21 to the aminoacid sequence of residue 235.

7. the antibody of claim 1, it comprises heavy chain and light chain, wherein said heavy chain forming from residue 20 to the aminoacid sequence of residue 467 by SEQ ID NO:2, and wherein said light chain forming from residue 21 to the aminoacid sequence of residue 235 by SEQ ID NO:4.

8. the antibody be separated, it produces by cultivating the mammalian host cell comprising the first polynucleotide and the second polynucleotide, wherein said first polynucleotide encoding heavy chain and the second polynucleotide encoding light chain, wherein:

A) described heavy chain comprises the variable region shown in the aminoacid sequence of SEQ ID NO:13; And

B) described light chain comprises the variable region shown in the aminoacid sequence of SEQ ID NO:14; And

Wherein said antibodies osteoprotegerin ligand (OPGL) and suppress the combination of OPGL and differentiation of osteoclast and activated receptor (ODAR).

9. the antibody of claim 8, wherein the first polynucleotide encoding comprises the heavy chain of the aminoacid sequence of SEQ ID NO:2, and the second polynucleotide encoding comprises the light chain of the aminoacid sequence of SEQ ID NO:4.

10. the antibody of claim 8, the wherein heavy chain that is made up of the aminoacid sequence of SEQ ID NO:2 of the first polynucleotide encoding, the light chain that the second polynucleotide encoding is made up of the aminoacid sequence of SEQ ID NO:4.

The antibody of 11. claims 8, wherein the first polynucleotide encoding comprise SEQ ID NO:2 from residue 20 to the heavy chain of the aminoacid sequence of residue 467, and wherein the second polynucleotide encoding comprise SEQ ID NO:4 from residue 21 to the light chain of the aminoacid sequence of residue 235.

The antibody of 12. claims 11, wherein the first polynucleotide encoding by SEQ ID NO:2 from residue 20 to the heavy chain that the aminoacid sequence of residue 467 forms, and wherein the second polynucleotide encoding by SEQ ID NO:4 from residue 21 to the light chain that the aminoacid sequence of residue 235 forms.

Antibody any one of 13. claim 8-12, wherein said first and second polynucleotide are parts of nucleic acid molecule separately.

14. 1 kinds of antibody, comprise heavy chain and light chain,

Wherein said heavy chain comprises three CDRs of SEQ ID NO:13; and wherein said light chain comprises three CDRs of SEQ ID NO:14, and wherein said antibodies osteoprotegerin part (OPGL) and suppress the combination of OPGL and differentiation of osteoclast and activated receptor (ODAR).

15. 1 kinds of antibody, comprise heavy chain and light chain,

Wherein said heavy chain comprises variable region and the constant region of the SEQ ID NO:2 with a carboxyl-terminus amino acid disappearance; Wherein said light chain comprises variable region and the constant region of SEQ ID NO:4;

And wherein said antibodies osteoprotegerin part (OPGL) and suppress the combination of OPGL and differentiation of osteoclast and activated receptor (ODAR).

16. claims 1,2,6, antibody any one of 9-12 and 14-15, wherein said heavy chain and described light chain form single-chain antibody.

The antibody of 17. claims 4, wherein said heavy chain and described light chain form single-chain antibody.

The antibody of 18. claims 16, it is single-chain Fv antibody.

19. claims 1,2,6, antibody any one of 9-12 and 14-15, wherein said antibody is Fab, Fab' or (Fab') ₂.

The antibody of 20. claims 4, wherein said antibody is Fab, Fab' or (Fab') ₂.

21. claim 1-3, antibody any one of 8 and 14, wherein said antibody is people's antibody completely.

The antibody of 22. claims 13, wherein said antibody is people's antibody completely.

23. claim 1-3, antibody any one of 6 and 7, the combination of wherein said antibody suppression osteoprotegerin ligand (OPGL) and differentiation of osteoclast and activated receptor (ODAR).

The antibody of 24. claims 4, the combination of wherein said antibody suppression osteoprotegerin ligand (OPGL) and differentiation of osteoclast and activated receptor (ODAR).

The antibody of 25. claims 5, the combination of wherein said antibody suppression osteoprotegerin ligand (OPGL) and differentiation of osteoclast and activated receptor (ODAR).

26. pharmaceutical compositions containing antibody any one of claim 1-25.

Antibody any one of 27. claim 1-25 or the purposes of the pharmaceutical composition of claim 28 in the medicine for the preparation of the bone lesion in treatment patient.

The purposes of 28. claims 27, wherein said bone lesion is relevant at least one situation being selected from lower group: osteoporosis, osteitis deformans, osteomyelitis, hypercalcemia, osteopenia, osteonecrosis, inflammatory conditions, auto immune conditions, rheumatoid arthritis and cancer.

The purposes of 29. claims 28, wherein said cancer is selected from mastocarcinoma, prostate cancer, thyroid carcinoma, kidney, lung cancer, esophagus cancer, the rectum cancer, bladder cancer, cervical cancer, ovarian cancer, liver cancer, gastrointestinal cancer, multiple myeloma, lymphoma and Hokdkin disease.

Antibody any one of 30. claim 1-25 or the pharmaceutical composition of claim 26 are for the preparation of using together at least one in radiation and chemotherapy thus being used for the treatment of the purposes in the medicine of the bone lesion relevant with cancer.

The purposes of 31. claims 30, wherein said chemotherapy relates to and is selected from anthracycline by least one, taxol, tamoxifen, Zorubicin, the pharmaceutical treatment of 5 FU 5 fluorouracil and luteinizing hormone-releasing hormone (LHRH) antagonist.

The purposes of 32. claims 30 or 31, wherein said cancer is selected from mastocarcinoma, prostate cancer, thyroid carcinoma, kidney, lung cancer, esophagus cancer, the rectum cancer, bladder cancer, cervical cancer, ovarian cancer, liver cancer, gastrointestinal cancer, multiple myeloma, lymphoma and Hokdkin disease.

33. 1 kinds of compositions be separated, it comprises the first polynucleotide and the second polynucleotide, wherein said first polynucleotide encoding heavy chain and the second polynucleotide encoding light chain, and the antibody wherein any one of the first and second polynucleotide encoding claim 1-25.

34. the composition of claim 33, wherein the first polynucleotide encoding comprises the heavy chain of the aminoacid sequence of SEQ ID NO:13, and the second polynucleotide encoding comprises the light chain of the aminoacid sequence of SEQ ID NO:14.

35. the composition of claim 33, wherein the first polynucleotide encoding comprises the heavy chain of the aminoacid sequence of SEQ ID NO:2, and the second polynucleotide encoding comprises the light chain of the aminoacid sequence of SEQ ID NO:4.

The composition of 36. claims 33, the wherein heavy chain that is made up of the aminoacid sequence of SEQ ID NO:2 of the first polynucleotide encoding, the light chain that the second polynucleotide encoding is made up of the aminoacid sequence of SEQ ID NO:4.

The composition of 37. claims 33, wherein the first polynucleotide encoding comprise SEQ ID NO:2 from residue 20 to the heavy chain of the aminoacid sequence of residue 467, the second polynucleotide encoding comprise SEQ ID NO:4 from residue 21 to the light chain of the aminoacid sequence of residue 235.

The composition of 38. claims 33, wherein the first polynucleotide encoding by SEQ ID NO:2 from residue 20 to the heavy chain that the aminoacid sequence of residue 467 forms, the second polynucleotide encoding by SEQ ID NO:4 from residue 21 to the light chain that the aminoacid sequence of residue 235 forms.

The composition of 39. claims 33, wherein the first polynucleotide comprise the nucleotide sequence of SEQ ID NO:1, and the second polynucleotide comprise the nucleotide sequence of SEQ ID NO:3.

The composition of any one of 40. claim 33-39, wherein said first and second polynucleotide are parts of identical nucleic acid molecule.

The composition of any one of 41. claim 33-39, wherein said first and second polynucleotide are parts of nucleic acid molecule separately.

The composition of 42. claims 40, wherein said nucleic acid molecule is carrier.

The composition of 43. claims 41, wherein said first polynucleotide are parts of the first carrier, and the second polynucleotide are parts of Second support.

The composition of 44. claims 42, wherein said carrier is virus vector.

The composition of 45. claims 43, at least one wherein in the first carrier and Second support is virus vector.

46. host cells be separated, it comprises the composition of any one of claim 33-45.

The host cell of 47. claims 46, it is prokaryotic host cell or eukaryotic host cell.

The host cell of 48. claims 47, it is mammalian host cell.

The host cell of 49. claims 48, wherein said host cell is selected from Chinese hamster ovary cell, HeLa cell, little hamster kidney cell, monkey-kidney cells and human liver cell cancer cells.

50. produce the method with osteoprotegerin ligand (OPGL) interactional antibody, and it comprises the host cell of any one of cultivating claim 46-49.

The method of the antibody of any one of 51. production claim 1-25, comprises and cultivates host cell and be separated described antibody.